GPCR Fusion Protein Containing an N-Terminal Autonomously Folding Stable Domain, and Crystals of the Same

Abstract

Certain embodiments provide a GPCR fusion protein. In particular embodiments, the GPCR fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions. In certain embodiments, the GPCR fusion protein may be crystallizable in a complex with a G-protein or in a complex with an antibody that binds to the IC3 loop of the GPCR.

Description

CROSS-REFERENCING

[0001] This application claims the benefit of U.S. provisional application Ser. Nos. 61/453,020, filed Mar. 15, 2011 and 61/507,425, filed Jul. 13, 2011, which are incorporated by reference in their entirety.

BACKGROUND

[0003] G protein-coupled receptor (GPCR) signaling plays a vital role in a number of physiological contexts including, but not limited to, metabolism, inflammation, neuronal function, and cardiovascular function. For instance, GPCRs include receptors for biogenic amines, e.g., dopamine, epinephrine, histamine, glutamate, acetylcholine, and serotonin; for purines such as ADP and ATP; for the vitamin niacin; for lipid mediators of inflammation such as prostaglandins, lipoxins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, follicle stimulating hormone, gonadotropin releasing hormone, ghrelin, motilin, neurokinin, and oxytocin; for non-hormone peptides such as beta-endorphin, dynorphin A, Leu-enkephalin, and Met-enkephalin; for the non-peptide hormone melatonin; for polypeptides such as C5a anaphylatoxin and chemokines; for proteases such as thrombin, trypsin, and factor Xa; and for sensory signal mediators, e.g., retinal photopigments and olfactory stimulatory molecules. GPCRs are of immense interest for drug development.

SUMMARY

[0004] A GPCR fusion protein is provided. In certain embodiments, the GPCR fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions. In certain embodiments, the GPCR fusion protein may be crystallizable in a complex with a G-protein or in a complex with an antibody that binds to the IC3 loop of the GPCR.

[0005] In particular embodiments, the GPCR fusion protein may further comprise an epitope tag N-terminal to the autonomously folding stable domain. In some cases, the GPCR fusion protein may further comprise a protease cleavage site between the epitope tag and the autonomously folding stable domain, thereby allowing the epitope tag to cleaved off.

[0006] In particular embodiments, the autonomously folding stable domain may comprises the amino acid sequence of lysozyme. In some cases, the GPCR fusion protein may also comprise a second autonomously folding stable domain between the TM5 and TM6 regions of the GPCR (i.e., in the IC3 loop of the GPCR).

[0007] In certain embodiments, the GPCR of the fusion protein may be active. The GPCR of the fusion protein may be naturally occurring or non-naturally occurring.

[0008] Also provided is a composition of matter comprising: a) a subject GPCR fusion protein; and b) a moiety complexed with the GPCR fusion protein. The moiety complexed with the GPCR fusion protein may be, for example, a G-protein or an antibody that is bound to the IC3 loop of the GPCR. The moiety may also be a ligand for the GPCR.

[0009] A nucleic acid encoding the subject GPCR fusion protein is also provided. In particular embodiments, the nucleic acid may encode, from 5' to 3': a) a signal sequence; b) an epitope tag; c) a protease cleavage site; d) an autonomously folding stable domain; and e) a GPCR. Also provided is a cell containing the nucleic acid. In particular cases, the fusion protein may be expressed in the cell, and disposed on the plasma membrane of the cell.

[0010] Also provided is a crystal comprising a crystalline form of the subject GPCR fusion protein. The crystal may further contain, for example, a G protein complexed with the GPCR fusion protein, a ligand for the GPCR, or an antibody that is bound to the IC3 loop of the GPCR. In particular embodiments, the crystallized GPCR fusion protein may comprise a second autonomously folding stable domain between the TM5 and TM6 regions of the GPCR.

[0011] Also provided is a method for producing the subject fusion protein. In some embodiments, this method may involve culturing the above-described cell to produce the GPCR fusion protein; and isolating the GPCR fusion protein from the cell. The may further comprises crystallizing the GPCR fusion protein to make crystals, e.g., using a bicelle crystallization method or a lipidic cubic phase crystallization method. Prior to crystallization, the isolated GPCR fusion protein may be combined with a moiety to which it complexes, e.g., the G protein to which it couples, a ligand or an antibody, for example, to produce a complexes. This method may further comprise obtaining atomic coordinates of the GPCR fusion protein from said crystal.

[0012] A method of determining a crystal structure is also provided. In certain cases this method comprises: receiving a subject GPCR fusion protein, crystallizing the fusion protein to produce a crystal; and obtaining atomic coordinates of the fusion protein from the crystal. Other embodiment include forwarding a subject GPCR fusion protein to a remote location, and receiving atomic coordinates for said GPCR fusion protein.

[0013] In particular embodiments, a composition comprising a fusion protein in crystalline form is provided in which the fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) a lysozyme domain, where the lysozyme domain is N-terminal to the GPCR.

[0014] In particular embodiments, the GPCR may comprise the amino acid sequence of a naturally occurring GPCR. In other embodiments, GPCR may comprise the amino acid sequence of a non-naturally occurring GPCR.

[0015] The domain, in certain cases, may comprise an amino acid sequence having at least 80% identity to the amino acid sequence of a wild-type lysozyme. For example, in certain cases, the domain may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of T4 lysozyme.

[0016] In particular embodiments, the GPCR may be a family A GPCR, a family B GPCR or a family C GCPR. In particular embodiments, the GPCR may be a receptor for a biogenic amine, a dopamine receptor, a seratonin receptor, an adrenergic receptor, a .beta.2-adrenergic receptor, a melanocortin receptor subtype 4, a ghrelin receptor, a metabotropic glutamate receptor or a chemokine receptor. The crystallized GPCR fusion protein may comprise a second autonomously folding stable domain (e.g., another lysozyme domain) between the TM5 and TM6 regions of the GPCR.

[0017] In some embodiments, the fusion protein is bound to a ligand for the GPCR. In particular embodiments, the fusion protein may be co-crystalized with a G protein to which the GPCR couples (which may be composed of the G.alpha., .beta. and .gamma. subunits) or an antibody that binds the IC3 loop of the GPCR, for example.

[0018] In particular cases, a GPCR-G-protein complex may be crystallized in conjunction with an antibody that stabilizes the G-protein in the same way as the nanobody described below. Such an antibody may be from any species and, in certain cases, may be a single chain antibody.

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1 is a schematic illustration of a GPCR, showing the canonical transmembrane regions (TM1, TM2, TM3, TM4, TM5, TM6, and TM7), intracellular regions (IC1, IC2, and IC3), and extracellular regions (EC1, EC2, and EC3).

[0020] FIG. 2 is a schematic illustration of a subject fusion protein, showing an autonomously folding stable domain that is N-terminal to a GPCR.

[0021] FIG. 3 is a schematic illustration of the fusion protein encoded by a subject nucleic acid. The encoded fusion protein contains an autonomously folding stable domain that is N-terminal to a GPCR. The protein further contains a signal sequence, an epitope tag and a protease cleavage site.

[0022] FIG. 4 shows exemplary sequences that may be employed in place of the lysozyme sequences of FIG. 5. From top to bottom, SEQ ID NOS: 2-6.

[0023] FIG. 5 shows the amino acid sequence of an exemplary fusion protein. SEQ ID NO:1. The HA signal peptide is shown in unbolded italic letters; the FLAG epitope tag is shown in underlined letters; the TEV recognition sequence is marked with non-underlined bold letters and the cleavage site is shown in asterisk. The full length T4L is shown by bold underlined letters and the .beta..sub.2AR sequence from Asp29 to Gly365 is shown by bold, underlined, italicized letters.

[0024] FIG. 6 shows the amino acid sequences of further exemplary fusion proteins. SEQ ID NOS: 7-13. The HA signal peptide is shown in unbolded italic letters; the FLAG epitope tag is shown in underlined letters; the TEV recognition sequence is marked with non-underlined bold letters and the cleavage site is shown in asterisk. The full length T4L is shown by bold underlined letters and the GPCR sequence is shown by bold, underlined, italicized letters.

[0025] FIG. 7. G protein cycle for the .beta..sub.2AR-Gs complex. a, Extracellular agonist binding to the .beta..sub.2AR leads to conformational rearrangements of the cytoplasmic ends of transmembrane segments that enable the G.sub.s heterotrimer (.alpha., .beta., and .gamma.) to bind the receptor. GDP is released from the .alpha. subunit upon formation of R:G complex. The GTP binds to the nucleotide-free .alpha. subunit resulting in dissociation of the .alpha. and .beta..gamma. subunits from the receptor. The subunits regulate their respective effector proteins adenylyl cyclase (AC) and Ca.sup.2+ channels. The G.sub.s heterotrimer reassembles from .alpha. and .beta..gamma. subunits following hydrolysis of GTP to GDP in the .alpha. subunit. b, The purified nucleotide-free .beta..sub.2AR-Gs protein complex maintained in detergent micelles. The Gs.alpha. subunit consists of two domains, the Ras domain (.alpha.Ras) and the .alpha.-helical domain (.alpha.AH). Both are involved in nucleotide binding. In the nucleotide-free state, the .alpha.AH domain has a variable position relative the .alpha.Ras domain.

[0026] FIG. 8. Overall structure of the .beta..sub.2AR Gs complex. a, Lattice packing of the complex shows alternating layers of receptor and G protein within the crystal. Abundant contacts are formed among proteins within the aqueous layers. b, The overall structure of the asymmetric unit contents shows the .beta..sub.2AR bound to an agonist (spheres) and engaged in extensive interactions with Gs.alpha.. G.alpha.s together with G.beta. and G.gamma. constitute the heterotrimeric G protein Gs. A Gs binding nanobody binds the G protein between the .alpha. and .beta. subunits. The nanobody (Nb35) facilitates crystallization, as does T4 lysozyme fused to the amino terminus of the .beta..sub.2AR. c, The biological complex omitting crystallization aids, showing its location and orientation within a cell membrane.

[0027] FIG. 9. Comparison of active and inactive .beta..sub.2AR structures. a, Side and cytoplasmic views of the .beta..sub.2AR-Gs structure compared to the inactive carazolol-bound .beta..sub.2AR structure (blue). Significant structural changes are seen for the intracellular domain of TM5 and TM6. TM5 is extended by two helical turns while TM6 is moved outward by 14 .ANG. as measured at the .alpha.-carbons of Glu268 in the two structures. b, .beta..sub.2AR-Gs compared with the nanobody-stabilized active state .beta..sub.2AR-Nb80 structure .sup.12c and d, The positions of residues in the E/DRY and NPxxY motifs and other key residues of the .beta..sub.2AR-Gs and .beta..sub.2AR-Nb80 structures as seen from the cytoplasmic side. All residues occupy very similar positions except Arg131 which in the .beta..sub.2AR-Nb80 structure interacts with the nanobody.

[0028] FIG. 10. Receptor-G protein interactions. a, b The .alpha.5-helix of G.alpha.s docks into a cavity formed on the intracellular side of the receptor by the opening of transmembrane helices 5 and 6. a. Within the transmembrane core, the interactions are primarily non-polar. An exception involves packing of Tyr391 of the .alpha.5-helix against Arg131 of the conserved DRY sequence in TM3 (see also FIG. 15). Arg131 also packs against Tyr of the conserved NPxxY sequence in TM7. b. As .alpha.5-helix exits the receptor it forms a network of polar interactions with TM5 and TM3. c, Receptor residues Thr68 and Asp 130 interact with the IL2 helix of the .beta..sub.2AR via Tyr141, positioning the helix so that Phe139 of the receptor docks into a hydrophobic pocket on the G protein surface, thereby structurally linking receptor-G protein interactions with the highly conserved DRY motif of the .beta..sub.2AR.

[0029] FIG. 11. Conformational changes in G.alpha.s. a, A comparison of Gs in the .beta..sub.2AR-Gs complex with the GTP.gamma.S-bound G.alpha.s (PDB ID: 1AZT). GTP.gamma.S is shown as spheres. The helical domain of G.alpha.s (G.alpha.sAH) exhibits a dramatic displacement relative to its position in the GTP.gamma.S-bound state. b, The .alpha.5-helix of G.alpha.s is rotated and displaced toward the .beta..sub.2AR, perturbing the .beta.6-.alpha.5 loop which otherwise forms part of the GTP.gamma.S binding pocket. c, The .beta.1-.alpha.1 loop (P-loop) and .beta.6-.alpha.5 loop of G.alpha.s interact with the phosphates and purine ring, respectively, of GTP.gamma.S in the GTP.gamma.S-Gs structure. d, The .beta.1-.alpha.1 and .beta.6-.alpha.5 loops are rearranged in the nucleotide-free .beta..sub.2AR-Gs structure.

[0030] FIG. 12. Proposed model for structural changes causing GDP release from the R:G complex. a, Alignment of the TM segments of .beta..sub.2AR in the .beta..sub.2AR-Gs structure and metarhodopsin II .sup.24(PDB ID: 3PQR) (purple) bound with the C-terminal peptide of transducin (blue). b, The C-terminal end of GsRas domain from the GTP.gamma.S bound Gs structure .sup.22 (PDB ID: 1AZT) is aligned with the C-terminal peptide of transducin. The C-terminal end of the .alpha.5-helix was moved away from the rest of the GsRas domain to avoid clashes with the .beta..sub.2AR. c, Cartoon of the .beta..sub.2AR-Gs peptide fusion construct used in the binding experiments (d). d, Competition binding experiments between [.sup.3H]-DHA and full agonist isoproterenol. Top panel shows binding data (reproduced from Rasmussen et al., 2011) on .beta..sub.2AR reconstituted in HDL particles with and without Gs heterotrimer. The fraction of .beta..sub.2AR in the K.sub.i high state for the .beta..sub.2AR with Gs is 0.55. Bottom panel shows binding to .beta..sub.2AR and a .beta..sub.2AR-G.alpha.s peptide fusion expressed in Sf9 cell membranes. The fraction of .beta..sub.2AR in the K.sub.i high state for the .beta..sub.2AR-G.alpha.s peptide fusion is 0.68. e, Same view as (b) but with metarhodopsin II structure and the C-terminal peptide removed. f, Comparison of GsRas domains of the transducin peptide aligned GTP.gamma.S bound Gs structure and the nucleotide-free Gs heterotrimer of the .beta..sub.2AR-Gs complex.

[0031] FIG. 13. Effect of nucleotide analogs, pH, and nanobodies on the stability of the R:G complex. a) Analytical gel filtration showing that nucleotides GDP and GTP.gamma.S (0.1 mM) cause dissociation of the R:G complex. b) The phosphates pyrophosphate and foscarnet (used at 5 mM) resemble the nucleotide phosphate groups, but do not cause disruption of the complex. When used as additives they improved crystal growth of both the T4L-.beta.2AR:Gs complex (without nanobodies), T4L-.beta.2AR:Gs:Nb37, and T4L-.beta.2AR:Gs:Nb35. c) The pH limit was determined to guide the preparation of crystallization screens. For the same purpose the effect of ionic strength (data not shown) was determined using NaCl at various concentrations. The complex is stable in 20, 100, and 500 mM but dissociates at 2.5 M NaCl. d) Nanobody 35 (Nb35, broken line) binds to the R:G complex (solid line) to form the R:G:Nb35 complex (red solid line) which is insensitive to GTP.degree. S treatment (solid line) in contrast to the treated R:G complex alone (broken line). Nb35 and Nb37 binds separate epitopes on the Gs heterotrimer to form a R:G:Nb35:Nb37 complex (solid line). Nb37 binding also prevents GTP.degree. S from dissociating the R:G complex (data not shown).

[0032] FIG. 14. Crystals of the T4L-.beta.2AR:Gs:Nb35 complex in sponge-like mesophase

[0033] FIG. 15. Views of electron density for residues in the R:G interface. a) The D/ERY motif at the cytoplasmic end of TM3. b) Packing interaction between Arg131 of the E/DRY motif and Tyr391 of C-terminal Gs.alpha.. c) The NPxxY in the cytoplasmic end of TM7. d) Interactions of Thr68 and Tyr141 with Asp130 of the E/DRY motif. Phe139 of IL2 is buried in a hydrophobic pocket in Gs.alpha.. e) The .beta.1-.alpha.1 loop (P-loop) of Gs.alpha. involved in nucleotide binding. Electron density maps are 2Fo-Fc maps contoured at 1 sigma.

[0034] FIG. 16. Flow-chart of the purification procedures for preparing R:G complex with Nb35

[0035] FIG. 17. Purity and homogeneity of the R:G complex: a) Analytical SDS-PAGE/Coomassie blue stain of samples obtained at various stages of receptor-G protein purification. BI167107 agonist bound, dephosphorylated, and deglycosylated receptor is used in excess of Gs heterotrimer for optimal coupling efficiency with the functional fraction of the G protein. Functional purification of Gs is archived through its interaction with the immobilized receptor on the M1 resin while non-functional/non-binding Gs is not retained. b) A representative elution profile of one of four consecutive preparative size exclusion chromatography (SEC) runs with fractionation indicated in red. SEC fractions containing the R:G complex (within the indicated dashed lines) were pooled, spin concentrated, and analyzed for purity and homogeneity by SDS-PAGE/Coomassie blue (a, lane 6), gel filtration (c), and by anion exchange chromotography (d). d) Upper panel shows elution pro le from an analytical ion exchange chromatography (IEC) run of .beta.2AR-365:Gs complex that was treated with .lamda. phosphatase prior to complex formation. Lower panel shows IEC of complex which was not dephosphorylated resulting in a heterogeneous preparation. Off-peak fractions from the preparative SEC (b) were used for analytical gel filtration experiments shown in FIGS. 13 and 21.

[0036] FIG. 18. Purification of Nb35 and determination of R:G:Nb mixing ratios a) Preparative ion exchange chromatography following nickel affinity chromatography purification of Nb35. The nanobody eluted in two populations (shown in red) as a minor peak and a major homogeneous peak which was collected, spin concentrated, and used for crystallography following determination of proper mixing ratio with the R:G complex as shown in (b). b) The R:G complex was mixed with slight excess of Nb35 (1 to 1.2 molar ratio of R:G complex to Nb35) on the basis of their protein concentrations and verified by analytical gel filtration.

[0037] FIG. 19. Formation of a stable R:G complex. A stable complex was achieved by the combined effects of: 1) binding a high affinity agonist to the receptor with an extremely slow dissociation rate (as described in Rasmussen et al., 2011); 2) formation of a nucleotide free complex in the presence of apyrase that hydrolyses released GDP preventing it from rebinding and causing a less stable R:G interaction; and 3) detergent exchange of DDM for MNG-3 that stabilizes the complex.

[0038] FIG. 20. Stabilizing effect of MNG-3 on the R:G complexes a) Analytical gel filtration of R:G complexes purified in DDM (in black), MNG-3 (in blue), or two MNG-3 analogs (in red and green) following incubation for 48 hrs at 4.degree. C. In contrast to DDM, the R:G complexes are stable in the MNG detergents. b) Effect of diluting unliganded purified .beta.2AR in either DDM or MNG-3 below the critical micelle concentration (CMC) of the detergent. Functional activity of the receptor was determined by 3H-dihydro alprenolol (3H-DHA) saturation binding. Diluting 2AR maintained in DDM by 1000-fold below the CMC cause loss in 3H-DHA binding (black data points) after 20 sec. In contrast, .beta.2AR in MNG-3 diluted 1000-fold below the CMC maintained full ability to bind 3H-DHA after 24 hrs.

[0039] FIG. 21. Effect of alkylating and reducing agents on the stability and aggregation of the R:G complex. a) Disulfide-mediated aggregation of the R:G complex was observed by size exclusion chromatography (SEC) following incubation at 0.degree. C. for 7 days in buffer containing 0.1 mM tris(2-carboxyethyl)phosphine (TCEP). b) Treatment of the complex with iodoacetamide (5 mM for 20 hrs at 20.degree. C.) led to dissociation of the complex. Alkylating free cysteines with iodoacetic acid and cadmium chloride also led to dissociation. c) Disulfide-mediated aggregation of the complex could be prevented by higher concentrations of reducing agents. Shown are the effects of 0.1, 1, and 10 mM TCEP for 1 hr at 20.degree. C., or 10 mM betamercaptoethanol (.beta.-ME, 1 hr at 20.degree. C.). Crystallization setups were performed using 1 to 5 mM TCEP, which was essential for optimal crystal growth.

[0040] FIG. 22. a. shows a schematic diagram of T4L-.beta..sub.2AR-.DELTA.ICL3 fusion protein used for crystallography, in including the .beta..sub.2AR residues, the wild type .beta..sub.2AR sequence, the HA signal peptide, the FLAG tag, the TEV recognition site the M96T, M98T mutations, the cysteines involved in disulfide bonds, disulfide bond linkages, the N187E mutation, and the 2-Ala linker. b. shows a chematic diagram of all of the T4L-.beta..sub.2AR-.DELTA.ICL3 constructs that were generated and evaluated for expression of functional receptor protein in insect cells. SEQ ID NOS: 18-29.

[0041] FIG. 23. a, b. Packing interactions mediated by T4L. Each T4L packs against three adjacent T4L-.beta..sub.2AR-.DELTA.ICL3 molecules and is involved in 4 packing interactions. The T4L and .beta..sub.2AR-.DELTA.-ICL3 from the reference molecule are shown. The T4L and .beta..sub.2AR-.DELTA.-ICL3 from the three adjacent molecules are shown. c-f. Close-up few of packing interactions 1-4. The residues involved in interactions are shown as spheres c. In interaction 1 the reference T4L packs against ECL2 of its fused .beta..sub.2AR-.DELTA.-ICL3. d. In interaction 2 the reference T4L packs against T4L of an adjacent T4L-.beta..sub.2AR-.DELTA.-ICL3. e. In interaction 3 the reference T4L packs against T4L, ECL2 and ECL3 of a second adjacent T4L-.beta..sub.2AR-.DELTA.-ICL3. f. In interaction 4 the reference T4L packs against ICL3 and helix 8 of a third T4L-.beta..sub.2AR-.DELTA.-ICL3.

[0042] FIG. 24. a. The crystal structure of the .beta..sub.2AR-Gs complex. The T4L, .beta..sub.2AR and the G-protein heterotrimer are shown in grey, as is the stabilizing nanobody. There is no packing interaction between the T4L and its fused .beta..sub.2AR. b. The crystal structure of T4L-.beta..sub.2AR-.DELTA.ICL3. The T4L is shown in red and its fused .beta..sub.2AR-.DELTA.ICL3 is shown. In contrast to the .beta..sub.2AR-Gs complex structure, there are packing interactions between the T4L and its fused receptor .beta..sub.2AR-.DELTA.ICL3.

[0043] FIG. 25. a. Saturation binding curves for antagonist dihydroalprenolol (DHA) binding to T4L-.beta..sub.2AR-.DELTA.ICL3 and the wild type .beta..sub.2AR365. b. Competition binding curves for agonist isopreterenol binding to T4L-.beta..sub.2AR-.DELTA.ICL3 and the wild type .beta..sub.2AR365.

[0044] FIG. 26. 2Fo-Fc map around the 2-Ala linker between T4L and the .beta..sub.2AR. The main chain of the fusion junction is shown in sticks. The electron density is shown in green mesh (1.sigma.). The T4L and .beta..sub.2AR-.DELTA.ICL3 are shown in grey, as is the 2-Ala linker.

[0045] FIG. 27. a. The superposed structures of the T4L-.beta..sub.2AR-.DELTA.ICL3 and the .beta..sub.2AR-T4L (pdb 2RH1). The T4L-.beta..sub.2AR-.DELTA.ICL3 and the .beta..sub.2AR-T4L are shown in grey. b. The extracellular side view of the superposed structures. c. The intracellular side view of the superposed structures. d. ICL2 in the .beta..sub.2AR-Fab5 structure (pdb 2R4R). e. ICL2 in the .beta..sub.2AR-T4L structure (pdb 2RH1). f. ICL2 in the T4L-.beta..sub.2AR-.DELTA.ICL3 structure. g. ICL2 in the structure of .beta..sub.2AR stabilized by Nb80 (pdb 3P0G) and h. ICL2 in the .beta..sub.2AR-Gs structure (pdb 3SN6)

[0046] FIG. 28. Shows a model of .beta.2AR bound to salmeterol, a partial agonist that is used to treat asthma. The partial-active state is stabilized by nanobody 71.

[0047] Certain of the figures described above are shown in color in U.S. provisional application Ser. Nos. 61/453,020, filed Mar. 15, 2011 and 61/507,425, filed Jul. 13, 2011. Those color figures, the brief description of those figures, and all references to color figures in those applications are incorporated by reference herein.

DEFINITIONS

[0048] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with general dictionaries of many of the terms used in this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0049] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0050] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0051] The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0052] "G-protein coupled receptors", or "GPCRs" are polypeptides that share a common structural motif, having seven regions of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans a membrane. As illustrated in FIG. 1, each span is identified by number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are joined by regions of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or "extracellular" side, of the cell membrane, referred to as "extracellular" regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. The transmembrane helices are also joined by regions of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular" side, of the cell membrane, referred to as "intracellular" regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The "carboxy" ("C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor lies in the extracellular space outside of the cell. GPCR structure and classification is generally well known in the art, and further discussion of GPCRs may be found in Probst, DNA Cell Biol. 1992 11:1-20; Marchese et al Genomics 23: 609-618, 1994; and the following books: Jurgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors published by Wiley-Liss (1st edition; Oct. 15, 1999); Kevin R. Lynch (Ed) Identification and Expression of G Protein-Coupled Receptors published by John Wiley & Sons (March 1998) and Tatsuya Haga (Ed), G Protein-Coupled Receptors, published by CRC Press (Sep. 24, 1999); and Steve Watson (Ed) G-Protein Linked Receptor Factsbook, published by Academic Press (1st edition; 1994). A schematic representation of a typical GPCR is shown in FIG. 1.

[0053] The term "naturally-occurring" in reference to a GPCR means a GPCR that is naturally produced (for example and not limitation, by a mammal or by a human). Such GPCRs are found in nature. The term "non-naturally occurring" in reference to a GPCR means a GPCR that is not naturally-occurring. Wild-type GPCRs that have been made constitutively active through mutation, and variants of naturally-occurring GPCRs, e.g., epitope-tagged GPCR and GPCRs lacking their native N-terminus are examples of non-naturally occurring GPCRs. Non-naturally occurring versions of a naturally occurring GPCR are activated by the same ligand as the naturally-occurring GPCR.

[0054] The term "ligand" means a molecule that specifically binds to a GPCR. A ligand may be, for example a polypeptide, a lipid, a small molecule, an antibody. A "native ligand" is a ligand that is an endogenous, natural ligand for a native GPCR. A ligand may be a GPCR "antagonist", "agonist", "partial agonist" or "inverse agonist", or the like.

[0055] A "modulator" is a ligand that increases or decreases a GPCR intracellular response when it is in contact with, e.g., binds, to a GPCR that is expressed in a cell. This term includes agonists, including partial agonists and inverse agonists, and antagonists.

[0056] A "deletion" is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental GPCR polypeptide or nucleic acid. In the context of a GPCR or a fragment thereof, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A GPCR or a fragment thereof may contain more than one deletion.

[0057] An "insertion" or "addition" is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental GPCR. "Insertion" generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while "addition" can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. In the context of a GPCR or fragment thereof, an insertion or addition is usually of about 1, about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A GPCR or fragment thereof may contain more than one insertion. Reference to particular GPCR or group of GPCRs by name, e.g., reference to the serotonin or histamine receptor, is intended to refer to the wild type receptor as well as active variants of that receptor that can bind to the same ligand as the wild type receptor and/or transduce a signal in the same way as the wild type receptor.

[0058] A "substitution" results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental GPCR or a fragment thereof. It is understood that a GPCR or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on GPCR activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

[0059] The term "biologically active", with respect to a GPCR, refers to a GPCR having a biochemical function (e.g., a binding function, a signal transduction function, or an ability to change conformation as a result of ligand binding) of a naturally occurring GPCR.

[0060] As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations. Reference to an "amount" of a GPCR in these contexts is not intended to require quantitative assessment, and may be either qualitative or quantitative, unless specifically indicated otherwise.

[0061] The terms "polypeptide" and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0062] The term "fusion protein" or grammatical equivalents thereof is meant a protein composed of a plurality of polypeptide components, that while typically unjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, .beta.-galactosidase, luciferase, etc.; and the like.

[0063] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0064] The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab').sub.2, and or other antibody fragments that retain specific binding to antigen.

[0065] Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab').sub.2, as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., "Immunology", Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986),). This term also encompasses so-called "phage display" antibodies.

[0066] A "monovalent" antibody is an antibody that has a single antigen binding region. Fab fragments, scFv antibodies, and phage display antibodies are types of monovalent antibodies, although others are known. A "Fab" fragment of an antibody has a single binding region, and may be made by papain digestion of a full length monoclonal antibody. A single chain variable (or "scFv") fragment of an antibody is an antibody fragment containing the variable regions of the heavy and light chains of immunoglobulins, linked together with a short flexible linker.

[0067] As used herein the term "isolated," when used in the context of an isolated compound, refers to a compound of interest that is in an environment different from that in which the compound naturally occurs. "Isolated" is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.

[0068] As used herein, the term "substantially pure" refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, or at least 90% free from other components with which it is naturally associated.

[0069] A "coding sequence" or a sequence that "encodes" a selected polypeptide, is a nucleic acid molecule which can be transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in a host cell when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. Other "control elements" may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence.

[0070] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. In the case of a promoter, a promoter that is operably linked to a coding sequence will effect the expression of a coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0071] By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.

[0072] A "vector" is capable of transferring gene sequences to a host cell. Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to host cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.

[0073] An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassettes can be constructed into a "vector," "vector construct," "expression vector," or "gene transfer vector," in order to transfer the expression cassette into a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0074] A first polynucleotide is "derived from" or "corresponds to" a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above.

[0075] A first polypeptide is "derived from" or "corresponds to" a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.

[0076] The term "autonomously folding stable domain" is intended to exclude the amino acid sequence of a reporter protein, e.g., an optically detectable protein such as a fluorescent protein (e.g., GFP, CFP or YFP) or luciferase, and also excludes amino acid sequences that are at least 90% identical to the extracellular of a naturally occurring GPCR.

[0077] The term "active form" or "native state" of a protein is a protein that is folded in a way so as to be active. A GPCR is in its active form if it can bind ligand, alter conformation in response to ligand binding, and/or transduce a signal which may or may not be induced by ligand binding. An active or native protein is not denatured.

[0078] The term "stable domain" is a polypeptide domain that, when folded in its active form, is stable, i.e., does not readily become inactive or denatured.

[0079] The term "folds autonomously" indicates a protein that folds into its active form in a cell, without biochemical denaturation and renaturation of the protein, and without chaperones.

[0080] The term "naturally-occurring" refers to an object that is found in nature.

[0081] The term "non-naturally-occurring" refers to an object that is not found in nature.

[0082] The term "heterologous", in the context of two things that are heterologous to one another, refers to two things that do not exist in the same arrangement in nature.

[0083] The term "signal sequence" or "signal peptide" refers to a sequence of amino acids at the N-terminal portion of a protein, which facilitates the secretion of the mature form of the protein through the plasma membrane. The mature form of the protein lacks the signal sequence which is cleaved off during the secretion process.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0084] In the following description, the fusion protein is described first, followed by a discussion of the crystallization method in which the fusion protein may be employed.

Fusion Proteins

[0085] As noted above, a subject fusion protein comprise: a) GPCR; and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The autonomously folding stable domain is believed to provide a polar surface for crystal lattice contacts on the extracellular surface of the protein, thereby allowing the fusion protein to be crystallized. In particular embodiments, the protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions, although other crystallization conditions may be employed. A polar surface for crystal lattice contacts on the extracellular surface of the protein provides several options for crystallizing the fusion protein. In one embodiment, the fusion protein may be crystallized as a complex with the G-protein to which the GPCR couples. In another embodiment, the protein may be crystallized as a complex with an monovalent antibody that binds to the IC3 loop of the GPCR, as described in published US patent application US20090148510 and by Rasmusson et al (Nature 2007 450: 383-388), which publications are incorporated by reference for disclosure of those methods. In another embodiment, the third intracellular loop of the GPCR may contain another autonomously folding stable domain (which may be the same as or different to the autonomously folding stable domain at the N-terminal end of the protein) as described in Rosenbaum et al (Science 2007 318: 1266-73) and published U.S. patent application US20090118474, which publications are incorporated by reference for disclosure of those methods

[0086] In very general terms, such a fusion protein may be made by substituting the N-terminal extracellular region of a GPCR with an autonomously folding stable protein that is globular and readily crystallizable, e.g., lysozyme, chitinase, glucose isomerase, xylanase, trypsin inhibitor, crambin or ribonuclease, for example. During crystallization, the autonomously folding stable domain is thought to provides a polar surface for crystal lattice contacts on the extracellular surface of the protein, thereby facilitating crystallization of the protein.

[0087] As will be described in greater detail below, the GPCR fusion protein may be produced using a nucleic acid encoding a longer protein that, in order from N- to C-terminus, contains a signal peptide, an epitope tag and a protease cleavage site and the GPCR fusion protein. The longer protein is produced in the cell. During secretion, the signal peptide is cleaved from the protein and the resulting protein can be purified using the epitope tag. The epitope tag can be cleaved from the GPCR fusion protein prior use. Various signal peptides, epitope tags and protease cleavage sites and methods for their use are known in the art.

[0088] GPCRs

[0089] Any known GPCR is suitable for use in the subject method. A disclosure of the sequences and phylogenetic relationships between 277 GPCRs is provided in Joost et al. (Genome Biol. 2002 3:RESEARCH0063, the entire contents of which is incorporated by reference) and, as such, at least 277 GPCRs are suitable for the subject methods. A more recent disclosure of the sequences and phylogenetic relationships between 367 human and 392 mouse GPCRs is provided in Vassilatis et al. (Proc Natl Acad Sci 2003 100:4903-8 and www.primalinc.com, each of which is hereby incorporated by reference in its entirely) and, as such, at least 367 human and at least 392 mouse GPCRs are suitable for the subject methods. GPCR families are also described in Fredriksson et al (Mol. Pharmacol. 2003 63, 1256-72).

[0090] The methods may be used, by way of exemplification, for purinergic receptors, vitamin receptors, lipid receptors, peptide hormone receptors, non-hormone peptide receptors, non-peptide hormone receptors, polypeptide receptors, protease receptors, receptors for sensory signal mediator, and biogenic amine receptors not including .beta.2-adrenergic receptor. In certain embodiments, said biogenic amine receptor does not include an adrenoreceptor. .alpha.-type adrenoreceptors (e.g. .alpha..sub.1A, .alpha..sub.1B or .alpha..sub.1C adrenoreceptors), and .beta.-type adrenoreceptors (e.g. .beta..sub.1, .beta..sub.2, or .beta..sub.3 adrenoreceptors) are discussed in Singh et al., J. Cell Phys. 189:257-265, 2001.

[0091] It is recognized that both native (naturally occurring) and altered native (non-naturally occurring) GPCRs may be used in the subject methods. In certain embodiments, therefore, an altered native GPCR (e.g. a native GPCR that is altered by an amino acid substitution, deletion and/or insertion) such that it binds the same ligand as a corresponding native GPCR, and/or couples to a G-protein as a result of the binding. In certain cases, a GPCR employed herein may have an amino acid sequence that is at least 80% identical to, e.g., at least 90% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical, to at least the heptahelical domain of a naturally occurring GPCR. A GPCR employed herein may optionally contain the C-terminal domain of a GPCR. In certain embodiments, a native GPCR may be "trimmed back" from its N-terminus and/or its C-terminus to leave its heptahelical domain, prior to use.

[0092] As such, the following GPCRs (native or altered) find particular use as parental GPCRs in the subject methods: cholinergic receptor, muscarinic 3; melanin-concentrating hormone receptor 2; cholinergic receptor, muscarinic 4; niacin receptor; histamine 4 receptor; ghrelin receptor; CXCR3 chemokine receptor; motilin receptor; 5-hydroxytryptamine (serotonin) receptor 2A; 5-hydroxytryptamine (serotonin) receptor 2B; 5-hydroxytryptamine (serotonin) receptor 2C; dopamine receptor D3; dopamine receptor D4; dopamine receptor D1; histamine receptor H2; histamine receptor H3; galanin receptor 1; neuropeptide Y receptor Y1; angiotensin II receptor 1; neurotensin receptor 1; melanocortin 4 receptor; glucagon-like peptide 1 receptor; adenosine A1 receptor; cannabinoid receptor 1; and melanin-concentrating hormone receptor 1.

[0093] In particular embodiments, the GPCR may belong to one of the following GPCR families: amine, peptide, glycoprotein hormone, opsin, olfactory, prostanoid, nucleotide-like, cannabinoid, platelet activating factor, gonadotropin-releasing hormone, thyrotropin-releasing hormone or melatonin families, as defined by Lapinsh et al (Classification of G-protein coupled receptors by alignment-independent extraction of principle chemical properties of primary amino acid sequences. Prot. Sci. 2002 11:795-805). The subject GPCR may be a family A GPCR (rhodopsin-like), family B GPCR (secretin-like, which includes the PTH and glucagon receptors), or a family C GPCR (glutamate receptor-like, which includes the GABA glutamate receptors), or an "other" family GPCR (which includes adhesion, frizzled, taste type-2, and unclassified family members).

[0094] In the subject methods, the N-terminal extracellular region N-terminal to the TM1 region of a GPCR is usually identified, and replaced with an autonomously folding stable domain to produce a fusion protein. A schematic representation of the prototypical structure of a GPCR is provided in FIG. 1, where these regions, in the context of the entire structure of a GPCR, may be seen. A schematic representation of a subject fusion protein is shown in FIG. 2.

[0095] The N-terminal extracellular region is readily discernable by one of skill in the art using, for example, a program for identifying transmembrane regions: once transmembrane region TM1 is identified, the N-terminal extracellular region will be apparent. The N-terminal extracellular region may also be identified using such methods as pairwise or multiple sequence alignment (e.g. using the GAP or BESTFIT of the University of Wisconsin's GCG program, or CLUSTAL alignment programs, Higgins et al., Gene. 1988 73:237-44), using a target GPCR and, for example, GPCRs of known structure.

[0096] Suitable programs for identifying transmembrane regions include those described by Moller et al., (Bioinformatics, 17:646-653, 2001). A particularly suitable program is called "TMHMM" Krogh et al., (Journal of Molecular Biology, 305:567-580, 2001). To use these programs via a user interface, a sequence corresponding to a GPCR or a fragment thereof is entered into the user interface and the program run. Such programs are currently available over the world wide web, for example at the website of the Center for Biological Sequence Analysis at cbs.dtu.dk/services/. The output of these programs may be variable in terms its format, however they usually indicate transmembrane regions of a GPCR using amino acid coordinates of a GPCR.

[0097] When TM regions of a GPCR polypeptide are determined using TMHMM, the prototypical GPCR profile is usually obtained: an N-terminus that is extracellular, followed by a segment comprising seven TM regions, and further followed by a C-terminus that is intracellular. TM numbering for this prototypical GPCR profile begins with the most N-terminally disposed TM region (TM1) and concludes with the most C-terminally disposed TM region (TM7).

[0098] In certain cases, once the N-terminal extracellular region is identified in a GPCR, a suitable region of amino acids is chosen for substitution with the amino acid sequence of the autonomously folding stable domain. In certain embodiments, the C-terminus of the autonomously folding stable domain is linked to the amino acid that is within 50 residues (e.g., e.g., 1-5, 1-10, 1-20, 1-30, 1-40, etc. residues) N-terminal to the N-terminal amino acid of the TM 1 region of the GPCR, although linkages outside of this region are envisioned. In one exemplary embodiment, amino acids that are at the N-terminal end of the TM1 region (i.e., within what would be referred to as the TM1 region) may be replaced in addition the amino acids that are N-terminal to the TM region. In particular embodiments, this junction may be optimized to provide for maximal expression and receptor activity.

[0099] In addition to substituting N-terminal extracellular region of a GPCR with a autonomously folding stable domain, as described above, in certain cases, the intracellular C-terminal region of the GPCR (which may C-terminal to the cysteine palmitoylation site that is approximately 10 to 25 amino acid residues downstream of a conserved NPXXY motif), may be deleted. In certain cases, the 20-30 amino acids immediately C-terminal to the cysteine palmitoylation site are not deleted. In particular embodiments, this position may be optimized to provide for maximal expression and receptor activity.

[0100] Autonomously Folding Stable Domains

[0101] In particular embodiments, the autonomously folding stable domain is a polypeptide than can fold autonomously in a variety of cellular expression hosts, and is resistant to chemical and thermal denaturation. In particular embodiments, the autonomously folding stable domains may be derived from a protein that is known to be highly crystallizable in a variety of space groups and crystal packing arrangements. In certain cases, the stable, folded protein insertion may also shield the fusion protein from proteolysis, and may itself be protease resistant. Lysozyme is one such polypeptide, however many others are known.

[0102] In certain embodiments, a autonomously folding stable domain of a subject fusion protein may be a soluble, stable protein (e.g., a protein displaying resistance to thermal and chemical denaturation) that folds autonomously of the GPCR portion of the fusion protein, in a cell. In certain cases, the stable, autonomously folding stable domain may have no cysteine residues (or may be engineered to have no cysteine residues) in order to avoid potential disulphide bonds between the autonomously folding stable domain and a GPCR portion of the fusion protein, or internal disulphide bonds. Autonomously folding stable domains are conformationally restrained, and are resistant to protease cleavage.

[0103] In certain cases, the autonomously folding stable domain may contain most or all of the amino acid sequence of a polypeptide that is readily crystallized. Such proteins may be characterized by a large number of deposits in the protein data bank (www.rcsb.org) in a variety of space groups and crystal packing arrangements. While examples that employ lysozyme as stable, folded protein insertion are discussed below, the general principles may be used to employ any of a number of polypeptides that have the characteristics discussed above. Autonomously folding stable domain candidates include those containing the amino acid sequence of proteins that are readily crystallized including, but not limited to: lysozyme, chitinase, glucose isomerase, xylanase, trypsin inhibitor, crambin, ribonuclease. Other suitable polypeptides may be found at the BMCD database (Gilliland et al 1994. The Biological Macromolecule Crystallization Database, Version 3.0: New Features, Data, and the NASA Archive for Protein Crystal Growth Data. Acta Crystallogr. D50 408-413), as published to the world wide web.

[0104] In certain embodiments, the autonomously folding stable domain used may be at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical or at least 98% identical to a wild type protein. Many suitable wild type proteins, including non-naturally occurring variants thereof, are readily crystalizable.

[0105] In one embodiment, the autonomously folding stable domain may be of the lysozyme superfamily, which share a common structure and are readily crystallized. Such proteins are described in, e.g., Wohlkonig et al (Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs. PLoS ONE 2010 5: e15388).

[0106] As noted above, one such autonomously folding stable domain that may be employed in a subject fusion protein is lysozyme. Lysozyme is a highly crystallizable protein (see, e.g., Strynadka et al Lysozyme: a model enzyme in protein crystallography EXS 1996 75: 185-222) and at present over 200 atomic coordinates for various lysozymes, including many wild-type lysozymes and variants thereof, including lysozymes from phage T4, human, swan, rainbow trout, guinea fowl, soft-shelled turtle, tapes japonica, nurse shark, mouse sperm, dog and phage P1, as well as man-made variants thereof, have been deposited in NCBI's structure database. A subject fusion protein may contain any of a wide variety of lysozyme sequences. See, e.g., Strynadka et al (Lysozyme: a model enzyme in protein crystallography (EXS. 1996; 75:185-222), Evrard et al (Crystal structure of the lysozyme from bacteriophage lambda and its relationship with V and C-type lysozymes) J. Mol. Biol. 1998 276:151-64), Forsythe et al (Crystallization of chicken egg-white lysozyme from ammonium sulfate. Acta Crystallogr D Biol Crystallogr. 1997 53:795-7), Remington et al (Structure of the Lysozyme from Bacteriophage T4: An Electron Density Map at 2.4A Resolution), Lyne et al (Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis. J Biol Chem. 1990 265:6928-30), Marana et al. (Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 62:750-2), Harada et al (Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus. J Mol Biol. 1989 207:851-2) and Yao et al (Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme). J. Biochem. 1992 111:1-3).

[0107] The length of the autonomously folding stable domain may be in the range of 50-500 amino acids, e.g., 80-200 amino acids in length, although autonomously folding stable domain having lengths outside of this range are also envisioned.

[0108] As noted above, the autonomously folding stable domain is not fluorescent or light-emitting. As such, the autonomously folding stable domain is not CFP, GFP, YFP, luciferase, or other light emitting, fluorescent variants thereof. In certain cases, a autonomously folding stable domain does not contain a flexible linker (e.g., a flexible polyglycine linker) or other such conformationally unrestrained regions. In certain cases, the autonomously folding stable domain contains a sequence of amino acids from a protein that has a crystal structure that has been solved. In certain cases, the stable, folded protein insertion should not have highly flexible loop region characterized by high cyrstallographic temperature factors (i.e., high B-factors).

[0109] An exemplary amino acid sequence for exemplary lysozyme fusion protein is set forth in FIG. 5, and the amino acid sequences of exemplary alternative additions (which may be substituted into any of the sequences of FIG. 5 in place of the lysozyme sequence) are shown in FIG. 4. These sequences include the sequences of trypsin inhibitor, calbindin, barnase, xylanase, glucokinase or a cytochrome, e.g., cytochrome a, b or c, although other sequences can be readily used. In particular embodiments, any of the proteins listed in table 1 of Papandreou et al (Eur. J. Biochem. 271, 4762-4768 (2004) FEBS 2004) or any of the 674 globular proteins listed by Wang and Yuan (Proteins 2000 38, 165-175) (which publications are incorporated by reference for disclosure of individual proteins), including orthologs from other species and variants proteins that are at least 80% identical to the listed proteins. Exemplary sequences include those of apolipophorin-III, staphylococcal nuclease, RNAse sa, uteroglobin, xylanase II, glutaredoxin, myohemerythin, bacillus 1-3, 1-4-.beta.-glucanase, orotate phosphoribosyltransferase, cytochrome b562, serine esterase, fructose permease, subunit IIb, fibritin, legume lectin, chloramphenicol acetyltransferase, cytochrome c oxidase, adenovirus fibre, flavodoxin, phospholipase a2, stnv coat protein, signal transduction protein, lysin, pseudoazurin, cutinase, retinoid-x receptor .alpha., transthyretin, dihydropteridin reductase, cytochrome c3, picornavirus, ch-p21 ras, interleukin-10, cellular retinoic-acid-binding protein, retroviral integrase, catalytic domain, oncomodulin, 2 (hiv-2) protease, glutamate receptor ligand binding core, calcium-binding protein, histidine-containing phosphocarrier, cellulase e2, parvalbumin, ubiquitin, triosephosphate isomerase, myoglobin, 2fe-2s ferredoxin, endonuclease, glycera globin, lysozyme, goose, uracil-dna glycosylase, lamprey globin, lysozyme, chicken, lumazine synthase, hemoglobin (horse), profilin, hypothetical protein ybea, hemoglobin (human), ribosomal protein, d-tyr trnatyr deacylase, erythrocruorin, integrase, coagulation factor x, leukemia inhibitory factor, glycosylasparaginase, carboxypeptidase inhibitor, mitochondrial cytochrome c, astacin, mhc class II p41 invariantchain fragment, cytochrome c2, diphtheria toxin, methylamine dehydrogenase, phospholipase, nadh oxidase, ovomucoid iii domain, dna-binding protein, signal transduction protein, ldl receptor, pheromone, ferredoxin ii, peptostreptococcus, anti-platelet protein, phosphatidylinositol 3-kinase, ferredoxin ii, desulfovibrio gigas, crambin, .alpha.-spectrin, sh3 domain, 1c0ba ribonuclease a, heat-stable enterotoxin b, signal transduction protein, c-src tyrosine kinase, tgf-.beta.3, seed storage protein 7 s vicillin, prion protein domain, rubredoxin, clostridium pasteurianum, immunoglobulin, abrin a-chain, rubredoxin, archaeon pyrococcus furiosus, cd2, first domain, platelet factor 4, fasciculin, macromycin, chemokine (growth factor), plasminogen, cohesin-2 domain, (pro)cathepsin b, ectothiorhodospira vacuolata, glucose-specific factor iii, actinidin, hipip, allochromatium vinosum, staphylococcal nuclease, chymotrypsin inhibitor CI-2, collagen type VI, dna-binding protein, fk-506 binding, and factor IX.

[0110] The amino acid sequences of a variety of exemplary GPCR fusion proteins that can be employed herein are set forth in FIG. 6. Given these sequences, suitable fusion proteins could be designed using other GPCR.

Nucleic Acids

[0111] A nucleic acid comprising a nucleotide sequence encoding a subject fusion protein is also provided. A subject nucleic acid may be produced by any method. Since the genetic code and recombinant techniques for manipulating nucleic acid are known, the design and production of nucleic acids encoding a subject fusion protein is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.) methods are used.

[0112] For example, site directed mutagenesis and subcloning may be used to introduce/delete/substitute nucleic acid residues in a polynucleotide encoding GPCR. In other embodiments, PCR may be used. Nucleic acids encoding a polypeptide of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297:1016-8).

[0113] In certain embodiments, the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e.g., human, species. Vectors comprising a subject nucleic acid are also provided. A vector may contain a subject nucleic acid, operably linked to a promoter.

[0114] A host cell (e.g., a host bacterial, mammalian, insect, plant or yeast cell) comprising a subject nucleic acid is also provided as well a culture of subject cells. The culture of cells may contain growth medium, as well as a population of the cells. The cells may be employed to make the subject fusion protein in a method that includes culturing the cells to provide for production of the fusion protein. In many embodiments, the fusion protein is directed to the plasma membrane of the cell, and is folded into its active form by the cell.

[0115] The native form of a subject fusion protein may be isolated from a subject cell by conventional technology, e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. For example, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215); ion-exchange chromatographic methods (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and Ellouz et al., (1987) Chromatography 396:307), including ion-exchange using materials with high resolution power (Medve et al., (1998) J. Chromatography A 808:153; hydrophobic interaction chromatography (Tomaz and Queiroz, (1999) J. Chromatography A 865:123; two-phase partitioning (Brumbauer, et al., (1999) Bioseparation 7:287); ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or size exclusion chromatography using, e.g., Sephadex G-75, may be employed.

[0116] In particular embodiments, the GPCR, e.g., the N- or C-terminus of the GPCR or an external loop of the GPCR, may be tagged with an affinity moiety, e.g., a his tag, GST, MBP, flag tag, or other antibody binding site, in order to facilitate purification of the GPCR fusion protein by affinity methods. Before crystallization, a subject fusion protein may be assayed to determine if the fusion protein is active, e.g., can bind ligand and change in conformation upon ligand binding, and if the fusion protein is resistant to protease cleavage. Such assays are well known in the art.

[0117] In particular embodiments and illustrated in FIG. 3, the protein encoded by the nucleic acid contains, from N-terminus to C-terminus: a) a signal sequence; b) an affinity, e.g., epitope, tag; c) a protease cleavage site; d) an autonomously folding stable domain; and e) a GPCR. During secretion, the signal peptide is cleaved from the protein and the resulting protein can be purified using the affinity tag. The affinity tag can be cleaved from the GPCR fusion protein prior use.

Crystallization Methods

[0118] Prior to crystallization, the isolated fusion protein may optionally be combined with a variety of moieties (e.g., an antibody (see, e.g., US20090148510, Rasmusson et al Nature 2007 450: 383-388 and Day et al Nature Methods 2007 4:927-9), a modulator (such as an agonist, an antagonist, a native ligand, etc., as described in, e.g., Rosenbaum Science. 2007 318:1266-73 etc), another GPCR, the G protein to which the GPCR couples or another protein, e.g., Gs, Gi, or Gq), that bind to the GPCR, to produce a complex. The complex is then crystallized and the atomic coordinates of the complex can be obtained.

[0119] A subject fusion protein may be crystallized using any of a variety of crystallization methods, many of which are reviewed in Caffrey Membrane protein crystallization. J Struct. Biol. 2003 142:108-32, including those that employ detergent micelles, bicelles and lipidic cubic phase (LCP). In general terms, the methods are lipid-based methods that include adding lipid to the fusion protein prior to crystallization. Such methods have previously been used to crystallize other membrane proteins. Many of these methods, including the lipidic cubic phase crystallization method and the bicelle crystallization method, exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method). Lipidic cubic phases crystallization methods are described in, for example: Landau et al, Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. 1996 93:14532-5; Gouaux, It's not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases. Structure. 1998 6:5-10; Rummel et al, Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins. J. Struct. Biol. 1998 121:82-91; and Nollert et al Lipidic cubic phases as matrices for membrane protein crystallization Methods. 2004 34:348-53, which publications are incorporated by reference for disclosure of those methods. Bicelle crystallization methods are described in, for example: Faham et al Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 2005 14:836-40. 2005 and Faham et al, Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol. 2002 Feb. 8; 316(1):1-6, which publications are incorporated by reference for disclosure of those methods.

[0120] Computer Models and Computer Systems

[0121] In certain embodiments, the above-described computer readable medium may further comprise programming for displaying a molecular model of a GPCR or a complex of the same crystalized by the instant method, programming for identifying a compound that binds to the GPCR and/or a database of structures of known test compounds, for example. A computer system comprising the computer-readable medium is also provided. The model may be displayed to a user via a display, e.g., a computer monitor, for example.

[0122] The atomic coordinates may be employed in conjunction with a modeling program to provide a model of the a GPCR or a complex of the same. As used herein, the term "model" refers to a representation in a tangible medium of the three dimensional structure of the a GPCR or a complex of the same. For example, a model can be a representation of the three dimensional structure in an electronic file, on a display, e.g., a computer screen, on a piece of paper (i.e., on a two dimensional medium), and/or as a ball-and-stick figure. Physical three-dimensional models are tangible and include, but are not limited to, stick models and space-filling models. The phrase "imaging the model on a computer screen" refers to the ability to express (or represent) and manipulate the model on a computer screen using appropriate computer hardware and software technology known to those skilled in the art. Such technology is available from a variety of sources including, for example, Evans and Sutherland, Salt Lake City, Utah, and Biosym Technologies, San Diego, Calif. The phrase "providing a picture of the model" refers to the ability to generate a "hard copy" of the model. Hard copies include both motion and still pictures. Computer screen images and pictures of the model can be visualized in a number of formats including space-filling representations, backbone traces, ribbon diagrams, and electron density maps. Exemplary modeling programs include, but are not limited to PYMOL, GRASP, or O software, for example.

[0123] In another embodiment, the invention provides a computer system having a memory comprising the above-described atomic coordinates; and a processor in communication with the memory, wherein the processor generates a molecular model having a three dimensional structure representative of a GPCR or a complex of the same. The processor can be adapted for identifying a candidate compound having a structure that is capable of binding to the a GPCR or a complex of the same, for example.

[0124] In the present disclosure, the processor may execute a modeling program which accesses data representative of the GPCR structure. In addition, the processor also can execute another program, a compound modeling program, which uses the three-dimensional model of the GPCR or a complex of the same to identify compounds having a chemical structure that binds to the GPCR or a complex of the same. In one embodiment the compound identification program and the structure modeling program are the same program. In another embodiment, the compound identification program and the structure modeling program are different programs, which programs may be stored on the same or different storage medium.

[0125] A number of exemplary public and commercial sources of libraries of compound structures are available, for example the Cambridge Structural Database (CSD), the Chemical Directory (ACD) from the company MDL (US), ZINC (Irwin and Shoichet, J. Chem. Inf Model. (2005) 45:177-82) as well as various electronic catalogues of publicly available compounds such as the National Cancer Institute (NCI, US) catalogue, ComGenex catalogue (Budapest, Hungary), and Asinex (Moscow, Russia). Such libraries may be used to allow computer-based docking of many compounds in order to identify those with potential to interact with the GPCR using the atomic coordinates described herein.

[0126] In certain cases, the method may further comprise a testing a compound to determine if it binds and/or modulates the GPCR or a complex of the same, using the atomic coordinates provided herein. In some embodiments, the method may further comprise obtaining the compound (e.g., purchasing or synthesizing the compound) and testing the compound to determine if it modulates (e.g., activates or inhibits) the GPCR, e.g., acts an agonist, antagonist or inverse agonist of the GPCR).

[0127] In some embodiments, the method employs a docking program that computationally tests known compounds for binding to the GPCR or complex of the same. Structural databases of known compounds are known in the art. In certain cases, compounds that are known to bind and modulate the GPCR or complex of the same may be computationally tested for binding to GPCR or complex of the same, e.g., in order to identify a binding site and/or facilitate the identification of active variants of an existing compound. Such compounds include compounds that are known to be agonists of the GPCR. In other cases, the method may include designing a compound that binds to the GPCR, either de novo, or by modifying an existing compound that is known to bind to the GPCR.

[0128] A method that comprises receiving a set of atomic coordinates for the GPCR or complex of the same; and identifying a compound that binds to said GPCR or complex of the same using the coordinates is also provided, as is a method comprising: forwarding to a remote location a set of atomic coordinates for the GPCR or complex of the same; and receiving the identity of a compound that binds to the GPCR or complex of the same.

[0129] In certain embodiments, a computer system comprising a memory comprising the atomic coordinates of a GPCR or complex of the same is provided. The atomic coordinates are useful as models for rationally identifying compounds that bind to the GPCR or complex of the same. Such compounds may be designed either de novo, or by modification of a known compound, for example. In other cases, binding compounds may be identified by testing known compounds to determine if the "dock" with a molecular model of the GPCR. Such docking methods are generally well known in the art.

[0130] The structure data provided can be used in conjunction with computer-modeling techniques to develop models of ligand-binding sites on the GPCR or complex of the same selected by analysis of the crystal structure data. The site models characterize the three-dimensional topography of site surface, as well as factors including van der Waals contacts, electrostatic interactions, and hydrogen-bonding opportunities. Computer simulation techniques are then used to map interaction positions for functional groups including but not limited to protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups may be designed into a candidate compound with the expectation that the candidate compound will specifically bind to the site.

[0131] The ability of a candidate compound to bind to a GPCR can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target with sufficient binding energy (i.e., binding energy corresponding to a dissociation constant with the target on the order of 10.sup.-2 M or tighter) may be synthesized and tested for their ability to bind to and modulate the GPCR. Such assays are known to those of skill in the art. The computational evaluation step thus avoids the unnecessary synthesis of compounds that are unlikely to bind the GPCR with adequate affinity.

[0132] A candidate compound may be computationally identified by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on the GPCR. One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with the GPCR, and more particularly with target sites on the GPCR. The process may begin by visual inspection of, for example a target site on a computer screen, based on the coordinates, or a subset of those coordinates. Selected fragments or chemical entities may then be positioned in a variety of orientations or "docked" within a target site of the GPCR as defined from analysis of the crystal structure data. Docking may be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.) and AMBER (University of California at San Francisco).

[0133] Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules," J. Med. Chem., 28, pp. 849-857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method," Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing," Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (Kunts, I. D., et al. "A Geometric Approach to Macromolecule-Ligand Interactions," J. Mol. Biol., 161, pp. 269-288 (1982)); DOCK is available from University of California, San Francisco, Calif.; CERIUS II (available from Molecular Simulations, Inc., San Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470-489 (1996)).

[0134] Also provided is a method of determining a crystal structure. This method may comprise receiving an above described fusion protein, crystallizing the fusion protein to produce a crystal; and obtaining atomic coordinates of the fusion protein from the crystal. The fusion protein may be received from a remote location (e.g., a different laboratory in the same building or campus, or from a different campus or city), and, in certain embodiments, the method may also comprise transmitting the atomic coordinates, e.g., by mail, e-mail or using the internet, to the remote location or to a third party.

[0135] In other embodiments, the method may comprise forwarding a fusion protein to a remote location where the protein may be crystallized and analyzed, and receiving the atomic coordinates of the fusion protein.

[0136] In some embodiments a method for displaying the three dimensional structure of a GPCR on a computer system is provided. This method may comprise: a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein the atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein the GPCR fusion protein is described above, b) modeling the atomic coordinates on the computer system using the modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR by; and c) displaying the model of the three dimensional structure on the computer system. The crystals also contain a ligand for the GPCR, and the method further comprises identifying the binding site for the ligand in the GPCR using the model. This method may further comprises identifying the amino acids in the binding site. This method may further comprise determining whether a test compound docks with the binding site using the model. This method may further comprise analyzing the packing between the test compound and surrounding amino acids in said binding site. In some embodiments, the analyzing may comprise calculating polar contacts between the ligand and the model.

[0137] In particular embodiments, a method for analyzing the three dimensional structure of a GPCR on a computer system is provided. This method may involve: a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein the atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein the GPCR fusion protein is described above, b) modeling the atomic coordinates on the computer system using the modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR.; and c) displaying the model of the three dimensional structure on the computer system. In certain cases, the crystals contain a ligand for the GPCR (e.g., a known inhibitor, natural ligand or agonist, etc.), and the method further comprises identifying the binding site for the ligand in the GPCR using the model. The analyzing step may comprise identifying amino acids that form polar contacts between the ligand and amino acids in the binding site, using the model. This method may further comprise determining whether a test compound, e.g., a candidate pharmaceutical, docks with the binding site using the model. The method may comprise analyzing the packing of the test compound and amino acids in the binding site, using the model. This method may further comprise making the modulator and testing it on the GPCR in the presence of a ligand for the GPCR.

[0138] In order to further illustrate the present invention, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.

Materials, Methods and Results I

[0139] Molecular Biology for the Generation of N-T4L Fused .beta.2AR Construct FLAAT

[0140] The previously generated construct .beta..sub.2AR365 was used as the template for further modification to generate the N-T4L fused .beta..sub.2AR construct FLAAT. In this .beta..sub.2AR365 template construct, the coding sequence of human .beta..sub.2AR encompassing Gly2 to Gly365 was cloned into the pFastbac1 Sf9 expression vector (Invitrogen). The HA signal peptide followed by FLAG epitope tag and tobacco etch virus (TEV) protease recognition sequence was directly added to the N-terminus of the receptor for expression and purification purpose. A point mutation of N187E was also introduced to the construct to disrupt this unwanted glycosylation site.

[0141] The DNA cassette encoding the full length T4L lysozyme (WT*, C54T, C97A) with 2 additional alanines attached at the C-terminus was made and amplified by PCR using previously described construct .beta..sub.2AR-T4L (Rasmussen et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007 450:383 and Cherezov et al High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007 318:1258-65) as the template and synthetic oligonucleotides as primers. This cassette was inserted into the .beta..sub.2AR365 construct between the end of the TEV protease recognition sequence and Asp29 of the receptor by using the Quickchange multi protocol (Stratagene). Two point mutations M96T, M98T were also introduced into the construct based on the Quickchange multi protocol using synthetic oligonucleotides as mutation primers. The protein sequence of the entire fusion FLAAT is shown in FIG. 5.

[0142] The entire FLAAT gene described above was further cloned into the Best-Bac Sf9 expression vector pv11393 (expressionsystems) using the restriction enzyme digestion site XbaI and EcoRI. The final construct was confirmed by NDA sequencing.

[0143] Expression and Purification of FLAAT from Baculovirus-Infected Sf9 Cells

[0144] Recombinant baculovirus was made from pv11393-FLAAT using Best-Bac expression system, as described by the system protocol (expressionsystem). FLAAT was expressed by Sf9 cells that were infected by this baculovirus with 1:50 dilution at the cell density of 4 million/ml. 1 .mu.M of receptor antagonist alprenolol was included to enhance the receptor stability and yield. The infected cells were harvested after 48 hs of incubation at 27.degree. C.

[0145] The harvested cells were lysed by vigorous stifling in 10 times volume of lysis buffer (10 mM TRIS-Cl pH 7.5, 2 mM EDTA) complemented with protease inhibitor Leupeptin (2.5 .mu.g/ml final concentration, Sigma) and Benzamindine (160 .mu.g/ml final concentration, Sigma) for 15 minutes. The FLAAT protein was extracted from the cell membrane by thorough homogenization using solubilization buffer (100 mM NaCl, 20 mM TRIS-Cl, pH 7.5, 1% Dodecylmaltoside) complemented with Leupeptin and Benzamindine (2.5 .mu.g/ml and 160 .mu.g/ml final concentration, respectively). 10 ml of solubilization buffer was used for each gram of cell pellet. The Dodecylmaltoside (DDM)-solubilized FLAAT bearing the FLAG epitope was then purified by M1 antibody affinity chromatography (Sigma). Extensive washing using HLS buffer (100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) was performed to get rid of alprenolol. The protein was then eluted with HLS buffer complemented with 5 mM EDTA, 200 .mu.g free FLAG peptide and saturating concentration of cholesterol hemisuccinate.

[0146] The eluted FLAAT was further purified by affinity chromatography using Sepharose attached with Alprenolol as previously described (Cherezov et al High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 2007 318:1258-65) in order to selectively isolate functional FLAAT from non-functional protein. HHS buffer (350 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) complemented with 300 .mu.M alprenolol and saturating concentration of cholesterol hemisuccinate was used to elute the protein. The eluted FLAAT bound with Alprenolol was then re-applied to M1 resin, allowing either washing off Alprenolol or exchanging Alprenolol with different ligand (for example, full agonist BI167107). Unliganded FLAAT or FLAAT bound with BI167107 was then eluted from M1 resin with HLS buffer complemented with 5 mM EDTA, 200 mg/ml free FLAG peptide and saturating concentration of cholesterol hemisuccinate. The FLAG epitope tag of FLAAT was removed by the treatment of tobacco etch virus (TEV) protease (invitrogen) for 3 hs at room temperature or overnight at 4.degree. C. The purity of the final FLAAT is more than 90% according to the result of SDS-PAGE electrophoresis.

[0147] Crystallization of the FLAAT-BI167107-NB80 Ternary Complex

[0148] Nanobody80 (NB80) was expressed and purified as previously described (Rasmussen Structure of a nanobody-stabilized active state of the .beta.(2) adrenoceptor. Nature. 2011 469:175-80.). The untagged FLAAT bound with high affinity agonist BI167107 was purified as described above. The purified FLAAT-BI167107 and NB80 was mixed with a 1:2 molar ratio. The FLAAT-BI167107-NB80 ternary complex was then isolated from free NB80 by size exclusion chromatography (SEC) using sephacryl S-200 column (GE health care life sciences) equilibrated in 100 mM NaCl, 10 mM HEPES pH 7.5, 0.1% DDM and 10 .mu.M BI167107. The same buffer was used as the running buffer for SEC.

[0149] The FLAAT-BI167107-NB80 complex after SEC was concentrated to a final concentration of 60 mg/ml using vivaspin concentrator (Sartorius-Stedim). The complex was crystallized using lipid cubic phase (LCP) method as previously described (Rosenbaum et al, GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science. 2007 318: 1266-73.). The protein complex was firstly mixed with lipid moloolein with a 1:1.5 mass ratio in room temperature. 0.1 .mu.l of the protein-lipid mixture drop was put in each well of a 24-well glass sandwich plate. The drop was then overlaid with 0.80 of precipitant and the well was sealed by glass coverslip. By using this method, the FLAAT-BI167107-NB80 ternary complex was crystallized in 31%-35% PEG400 (v/v) and 0.1M Tris-Cl, pH8.0 after 4 days of incubation in 20.degree. C.

Materials and Methods II

[0150] Expression and Purification of .beta.2AR, Gs Heterotrimer, and Nanobody-35

[0151] An N-terminally fused T4 lysozyme-.beta.2AR construct .beta.2AR truncated in position 365 (T4L-.beta.2AR, described in detail below) was expressed in Sf9 insect cell cultures infected with recombinant baculovirus (BestBac, Expression Systems), and solubilized in n-Dodecyl-.beta.-D-maltoside (DDM) according to methods described previously Kobilka (Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 1995 231, 269-271; see FIG. 16 for purification overview). A .beta.2AR construct truncated after residue 365 (.beta.2AR-365) was used for the majority of the analytical experiments and for deuterium exchange experiments. M1 Flag affinity chromatography (Sigma) served as the initial purification step followed by alprenolol-Sepharose chromatography for selection of functional receptor. A subsequent M1 Flag affinity chromatography step was used to exchange receptor-bound alprenolol for high-affinity agonist BI-167107. The agonist-bound receptor was eluted, dialyzed against buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM and 10 .mu.M BI-167107), treated with lambda phosphatase (New England Biolabs), and concentrated to approximately 50 mg ml.sup.-1 with a 50 kDa molecular weight cut off (MWCO) Millipore concentrator. Prior to spin concentration, the .beta.2AR-365 construct, but not T4L-.beta.2AR, was treated with PNGaseF (New England Biolabs) to remove amino-terminal N-linked glycosylation. The purified receptor was routinely analyzed by SDS-PAGE/Coomassie brilliant blue staining (see FIG. 17a).

[0152] Bovine G.alpha.s short, His6-bovine G.beta.1, and bovine G.gamma.2 were expressed in HighFive insect cells (Invitrogen) grown in Insect Xpress serum-free media (Lonza). Cultures were grown to a density of 1.5 million cells per ml and then infected with three separate Autographa californica nuclear polyhedrosis virus each containing the gene for one of the G protein subunits at a 1:1 multiplicity of infection (the viruses were a generous gift from Dr. Alfred Gilman). After 40-48 hours of incubation the infected cells were harvested by centrifugation and resuspended in 75 ml lysis buffer (50 mM HEPES, pH 8.0, 65 mM NaCl, 1.1 mM MgCl.sub.2, 1 mM EDTA, 1.times.PTT (35 .mu.g/ml phenylmethanesulfonyl fluoride, 32 .mu.g/ml tosyl phenylalanyl chloromethyl ketone, 32 .mu.g/ml tosyl lysyl chloromethyl ketone), 1.times. LS (3.2 .mu.g/ml leupeptin and 3.2 .mu.g/ml soybean trypsin inhibitor), 5 mM .beta.-mercaptoethanol (.beta.-ME), and 10 .mu.M GDP) per liter of culture volume. The suspension was pressurized with 600 psi N.sub.2 for 40 minutes in a nitrogen cavitation bomb (Parr Instrument Company). After depressurization, the lysate was centrifuged to remove nuclei and unlysed cells, and then ultracentrifuged at 180,000.times.g for 40 minutes. The pelleted membranes were resuspended in 30 ml wash buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 100 .mu.M MgCl.sub.2, 1.times.PTT, 1.times. LS, 5 mM .beta.-ME, 10 .mu.M GDP) per liter culture volume using a Dounce homogenizer and centrifuged again at 180,000.times.g for 40 minutes. The washed pellet was resuspended in a minimal volume of wash buffer and flash frozen with liquid nitrogen.

[0153] The frozen membranes were thawed and diluted to a total protein concentration of 5 mg/ml with fresh wash buffer. Sodium cholate detergent was added to the suspension at a final concentration of 1.0%, MgCl.sub.2 was added to a final concentration of 5 mM, and 0.05 mg of purified protein phosphatase 5 (prepared in house) was added per liter of culture volume. The sample was stirred on ice for 40 minutes, and then centrifuged at 180,000.times.g for 40 minutes to remove insoluble debris. The supernatant was diluted 5-fold with Ni-NTA load buffer (20 mM HEPES, pH 8.0, 363 mM NaCl, 1.25 mM MgCl.sub.2, 6.25 mM imidazole, 0.2% Anzergent 3-12, 1.times.PTT, 1.times. LS, 5 mM .beta.-ME, 10 .mu.M GDP), taking care to add the buffer slowly to avoid dropping the cholate concentration below its critical micelle concentration too quickly. 3 ml of Ni-NTA resin (Qiagen) pre-equlibrated in Ni-NTA wash buffer 1 (20 mM HEPES, pH 8.0, 300 mM NaCl, 2 mM MgCl.sub.2, 5 mM imidazole, 0.2% Cholate, 0.15% Anzergent 3-12, 1.times.PTT, 1.times.LS, 5 mM .beta.-ME, 10 .mu.M GDP) per liter culture volume was added and the sample was stirred on ice for 20 minutes. The resin was collected into a gravity column and washed with 4.times. column volumes of Ni-NTA wash buffer 1, Ni-NTA wash buffer 2 (20 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM MgCl.sub.2, 10 mM imidazole, 0.15% Anzergent 3-12, 0.1% DDM, 1.times.PTT, 1.times. LS, 5 mM .beta.-ME, 10 .mu.M GDP), and Ni-NTA wash buffer 3 (20 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM MgCl.sub.2, 5 mM imidazole, 0.1% DDM, 1.times.PTT, 1.times. LS, 5 mM .beta.-ME, 10 .mu.M GDP). The protein was eluted with Ni-NTA elution buffer (20 mM HEPES, pH 8.0, 40 mM NaCl, 1 mM MgCl2, 200 mM imidazole, 0.1% DDM, 1.times.PTT, 1.times. LS, 5 mM .beta.-ME, 10 .mu.M GDP). Protein-containing fractions were pooled and MnCl.sub.2 was added to a final concentration of 100 .mu.M. Fifty .mu.g of purified lambda protein phosphatase (prepared in house) was added per liter of culture volume and the elute was incubated on ice with stifling for 30 minutes. The eluate was passed through a 0.22 .mu.m filter and loaded directly onto a MonoQ HR 16/10 column (GE Healthcare) equilibrated in MonoQ buffer A (20 mM HEPES, pH 8.0, 50 mM NaCl, 100 .mu.M MgCl.sub.2, 0.1% DDM, 5 mM .beta.-ME, 1.times.PTT). The column was washed with 150 ml buffer A at 5 ml/min and bound proteins were eluted over 350 ml with a linear gradient up to 28% MonoQ buffer B (same as buffer A except with 1 M NaCl). Fractions were collected in tubes spotted with enough GDP to make a final concentration of 10 .mu.M. The Gs containing fractions were concentrated to 2 ml using a stirred ultrafiltration cell (Amicon) with a 10 kDa NMWL regenerated cellulose membrane (Millipore). The concentrated sample was run on a Superdex 200 prep grade XK 16/70 column (GE Healthcare) equilibrated in 5200 buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 1.1 mM MgCl.sub.2, 1 mM EDTA, 0.012% DDM, 100 .mu.M TCEP, 2 .mu.M GDP). The fractions containing pure Gs were pooled, glycerol was added to 10% final concentration, and then the protein was concentrated to at least 10 mg/ml using a 30 kDa MWCO centrifugal ultrafiltration device (Millipore). The concentrated sample was then aliquoted, flash frozen, and stored at -80.degree.. A typical yield of final, purified Gs heterotrimer from 8 liters of cell culture volume was 6 mg.

[0154] Nanobody-35 (Nb35) was expressed in the periplasm of E. coli strain WK6, extracted, and purified by nickel affinity chromatography according to previously described methods (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180) followed by ion-exchange chromatography (FIG. 18a) using a Mono S 10/100 GL column (GE Healthcare). Selected Nb35 fractions were dialysis against buffer (10 mM HEPES, pH 7.5, 100 mM NaCl) and concentrated to approximately 65 mg ml-1 with a 10 kDa MWCO Millipore concentrator.

[0155] Complex Formation, Stabilization and Purification

[0156] Formation of a stable complex (see FIG. 19) was accomplished by mixing Gs heterotrimer at approximately 100 .mu.M concentration with BI-167107 bound T4L-.beta..sub.2AR (or .beta.2AR-365) in molar excess (approximately 130 .mu.M) in 2 ml buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM EDTA, 3 mM MgCl.sub.2, 10 .mu.M BI-167107) and incubating for 3 hrs at room temperature. BI-167107, which was identified from screening and characterizing approximately 50 different .beta..sub.2AR agonists, has a dissociation half-time of approximately 30 hrs providing higher degree of stabilization to the active G protein-bound receptor than other full agonists such as isoproterenol (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180). To maintain the high-affinity nucleotide-free state of the complex, apyrase (25 mU/ml, NEB) was added after 90 min to hydrolyze residual GDP released from Gsupon binding to the receptor. GMP resulting from hydrolysis of GDP by apyrase has very poor affinity for the G protein in the complex. Rebinding of GDP can cause dissociation of the R:G complex (FIG. 13A).

[0157] The R:G complex in DDM shows significant dissociation after 48 hours at 4.degree. C. (FIG. 20A). Over 50 amphiphiles were screened and identified MNG-3 (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180; Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7, 1003-1008; NG-310, Affymetrix-Anatrace) and its closely related analogs as detergents that substantially stabilize the complex (FIGS. 20A and B). The complex was exchanged into MNG-3 by adding the R:G mixture (2 ml) to 8 ml buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 .mu.M BI-167107) containing 1% MNG-3 for 1 hr at room temperature.

[0158] At this stage the mixture contains the R:G complex, non-functional Gs, and an excess of .beta..sub.2AR. To separate functional R:G complex from non-functional Gs, and to complete the detergent exchange, the R:G complex was immobilized on M1 Flag resin and washed in buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 .mu.M BI-167107, and 3 mM CaCl.sub.2) containing 0.2% MNG-3. To prevent cysteine bridge-mediated aggregation of R:G complexes, 100 .mu.M TCEP was added to the eluted protein prior to concentrating it with a 50 kDa MWCO Millipore concentrator. Of note, it was discovered later that crystal growth improved at even higher TCEP concentrations (above 1 mM) compared to 100 .mu.M TCEP, and that the integrity of the R:G complex in MNG-3 was stable to 10 mM TCEP as measured by gel filtration analysis (FIG. 21C). In contrast, DDM-solubilized .beta..sub.2AR loses its ability to bind the high-affinity antagonist .sup.3H-dihydroalprenolol in 10 mM TCEP (data not shown), probably due to disruption of extracellular disulfide bonds. Iodoacetamide could not be used to block reactive cysteines on G.sub.s alpha and beta subunits as it caused dissociation of the R:G complex (fig. S9b). The final size exclusion chromatography procedure to separate excess free receptor from the R:G complex (FIG. 17b) was performed on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 0.02% MNG-3, 10 mM HEPES pH 7.5, 100 mM NaCl, 10 .mu.M BI-167107, and 100 .mu.M TCEP. Peak fractions were pooled (FIG. 17b) and concentrated to approximately 90 mg ml.sup.-1 with a 100 kDa MWCO Viva-spin concentrator and analyzed by SDS-PAGE/Coomassie brilliant blue staining (FIG. 17a) and gel filtration (FIG. 17c). To confirm a pure, homogeneous, and dephosphorylated preparation, the R:G complex was routinely analyzed by ion exchange chromatography (FIG. 17d).

[0159] Protein Engineering

[0160] To increase the probability of obtaining crystals of the R:G complex two strategies were used to increase the polar surface area on the extracellular side of the receptor. The first approach, to generate extracellular binding antibodies, was not successful. The second approach was to replace the flexible and presumably unstructured N-terminus with the globular protein T4 lysozyme (T4L) used previously to crystallize and solve the carazolol-bound receptor (Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 2007 318, 1266-1273). The construct used here (T4L-.beta..sub.2AR) contained the cleavable signal sequence followed by the M1 Flag epitope (DYKDDDDA; SEQ ID NO: 14), the TEV protease recognition sequence (ENLYFQG; SEQ ID NO: 15), bacteriophage T4 lysozyme from N2 through Y161 including C54T and C97A mutations, and a two residue alanine linker fused to the human .beta..sub.2AR sequence D29 through G365. The PNGaseF-inaccessible glycosylation site of the .beta..sub.2AR at N187 was mutated to Glu. M96 and M98 in the first extracellular loop were each replaced by Thr to increase the otherwise low expression level of T4L-.beta..sub.2AR. The threonine mutations did not affect ligand binding affinity for .sup.3H-dihydro-alprenolol, but caused a small, approximately two-fold decrease in affinity for isoproterenol.

[0161] The .beta..sub.2AR-Gs peptide fusion construct used for [.sup.3H]-DHA competition binding with isoproterenol was constructed from the receptor truncated at position 365 and fused to the last 21 amino acids of the G.alpha.s subunit (amino acids 374-394, except for C379A). A Gly-Ser is inserted between the receptor and the peptide. Also an extended TEV protease site (SENLYFQGS; SEQ ID NO: 16) was introduced in the .beta..sub.2AR between G360 and G361.

[0162] Stabilization of Gs with Nanobodies

[0163] From negative stain EM imaging, we observed that the alpha helical domain of G.alpha.s was flexible and therefore possibly responsible for poor crystal quality. Targeted stabilization of this domain was addressed by immunizing two llamas (Llama glama) with the bis(sulfosuccinimidyl)glutarate (BS2G, Pierce) cross-linked .beta..sub.2AR-Gs-BI-167107 ternary complex. Peripheral blood lymphocytes were isolated from the immunized animals to extract total RNA, prepare cDNA and construct a Nanobody phage display library according to published methods. Nb35 and Nb37 were enriched by two rounds of biopanning on the .beta..sub.2AR-Gs-BI-167107 ternary complex embedded in biotinylated high-density lipoprotein particles (Whorton, et al. Proc Natl Acad Sci USA 2007 104, 7682-7687). Nb35 and Nb37 were selected for further characterization because they bind the .beta..sub.2AR-Gs-BI-167107 ternary complex but not the free receptor in an ELISA assay. Nanobody binding to the R:G complex was confirmed by size exclusion chromatography (FIG. 13d), and it was noted that both nanobodies protected the complex from dissociation by GTP.gamma.S, suggestive of a stabilizing Gs:Nb interaction (FIG. 13d).

[0164] Crystallization

[0165] BI-167107 bound T4L-.beta..sub.2AR:Gs complex and Nb35 were mixed in 1:1.2 molar ratio. The small molar excess of Nb35 was verified by analytical gel filtration (see FIG. 15b). The mixture incubated for 1 hr at room temperature prior to mixing with 7.7 MAG containing 10% cholesterol (C8667, Sigma) in 1:1 protein to lipid ratio (w/w) using the twin-syringe mixing method reported previously. The concentration of R:G:Nb complex in 7.7 MAG was approximately 25 mg ml.sup.-1. The detergent MNG-3 may stabilize the T4L-.beta..sub.2AR-Gs complex during its incorporation into the lipid cubic phase. This may be due to the high affinity of MNG-3 for the receptor. The .beta..sub.2AR in MNG-3 maintains its structural integrity even when diluted below the CMC of the detergent, in contrast to .beta..sub.2AR in DDM, which rapidly loses binding activity (FIG. 20b). Moreover, MNG-3 improved crystal size and quality, as previously reported. The protein:lipid mixture was delivered through an LCP dispensing robot (Gryphon, Art Robbins Instruments) in 40 nl drops to either 24-well or 96-well glass sandwich plates and overlaid en-bloc with 0.8 .mu.l precipitant solution. Multiple crystallization leads were initially identified using in-house screens partly based on reagents from the StockOptions Salt kit (Hampton Research). Crystals for data collection were grown in 18 to 22% PEG 400, 100 mM MES pH 6.5 (FIG. 13c), 350 to 450 mM potassium nitrate, 10 mM foscarnet (FIG. 13b), 1 mM TCEP (FIG. 21c), and 10 .mu.M BI-167107 Crystals reached full size within 3-4 days at 20.degree. C. and were picked from a sponge-like mesophase and flash-frozen in liquid nitrogen without additional cryo-protectant.

[0166] Microcrystallography Data Collection and Processing.

[0167] Diffraction data were measured at the Advanced Photon Source beamline 23 ID-B. Hundreds of crystals were screened, and a final dataset was compiled using diffraction wedges of typically 10 degrees from 20 strongly diffracting crystals. All data reduction was performed using HKL2000 (Otwinowski. & Minor, W. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997 276, 307-326). Although in many cases diffraction to beyond 3 .ANG. was seen in initial frames, radiation damage and anisotropic diffraction resulted in low completeness in higher resolution shells. Analysis of the final dataset by the UCLA diffraction anisotropy server .sup.31 indicated that diffraction along the a* axis was superior to that in other directions. On the basis of an F/.sigma. (F) cutoff of 3 along each reciprocal space axis, reflections were subjected to an anisotropic truncation with resolution limits of 2.9, 3.2, and 3.2 Angstroms along a*, b*, and c* prior to use in refinement. The structure is reported to an overall resolution of 3.2 .ANG.. Despite the low completeness in the highest resolution shells (Table 3) inclusion of these reflections gave substantial improvements in map quality and lower Rfree during refinement.

[0168] Structure Solution and Refinement

[0169] The structure was solved by molecular replacement using Phaser. In order, the search models used were: the .beta. and .gamma. subunits from a Gi heterotrimer (PDB ID: 1GP2), the Gs alpha ras-like domain (PDB ID: 1AZT), the active-state .beta.2 adrenergic receptor (PDB ID: 3P0G), a .beta..sub.2AR binding nanobody (PDB ID: 3P0G), T4 lysozyme (PDB ID: 2RH1), and the Gs alpha helical domain (PDB ID: 1AZT). Following the determination of the initial structure by molecular replacement, rigid body refinement and simulated annealing were performed in Phenix and BUSTER, followed by restrained refinement and manual rebuilding in Coot. After iterative refinement and manual adjustments, the structure was refined in CNS using the DEN method. Although the resolution of this structure exceeds that for which DEN is typically most useful, the presence of several poorly resolved regions indicated that the incorporation of additional information to guide refinement could provide better results. The DEN reference models used were those used for molecular replacement, with the exception of NB35, which was well ordered and for which no higher resolution structure is available. Side chains were omitted from 52 residues for which there was no electron density past C.beta. below a low contour level of 0.7.sigma. in a 2Fo-Fc map. Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC.). MolProbity was used to determine Ramachandran statistics.

[0170] Competition Binding

[0171] Membranes expressing the .beta..sub.2AR or the .beta..sub.2AR-Gs peptide fusion were prepared from baculovirus-infected Sf9 cells and [.sup.3H]-dihydroalprenolol ([.sup.3H]-DHA) binding performed as previously described (Swaminath et al Mol Pharmacol 2002 61, 65-72). For competition binding, membranes were incubated with [.sup.3H]-DHA (1.1 nM final) and increasing concentrations of (-)-isoproterenol (ISO) for 1 hr before harvesting onto GF/B filters. Competition data were fitted to a two-site binding model and ISO high and low Ki's and fractions calculated using GraphPad prism.

Results II

Crystallization of the .beta.2AR-Gs Complex

[0172] One challenge for crystallogenesis was to prepare a stable .beta..sub.2AR-Gs complex in detergent solution. The .beta..sub.2AR and Gs couple efficiently in lipid bilayers, but not in detergents used to solubilize and purify these proteins. We found that a relatively stable .beta..sub.2AR-Gs complex could be prepared by mixing purified GDP-Gs (approximately 100 .mu.M final concentration) with a molar excess of purified .beta..sub.2AR bound to a high affinity agonist (BI-167107, Boehringer Ingelheim) in dodecylmaltoside solution. Apyrase, a non-selective purine pyrophosphatase, was added to hydrolyze GDP released from Gs on forming a complex with the .beta..sub.2AR. The complex was subsequently purified by sequential antibody affinity chromatography and size exclusion chromatography. The stability of the complex was enhanced by exchanging it into a recently developed maltose neopentyl glycol detergent (NG-310, Anatrace). The complex could be incubated at room temperature for 24 hrs without any noticeable degradation; however, initial efforts to crystallize the complex using sparse matrix screens in detergent micelles, bicelles and lipidic cubic phase (LCP) failed.

[0173] To further assess the quality of the complex, the protein was analyzed by single particle electron microscopy (EM). The results confirmed that the complex was monodispersed, and revealed two potential problems for obtaining diffraction of quality crystals. First, the detergent used to stabilize the complex formed a large micelle, leaving little polar surface on the extracellular side of the .beta..sub.2AR-Gs complex for the formation of crystal lattice contacts. The initial approach to this problem, which was to generate antibodies to the extracellular surface, was not successful. As an alternative approach, we replaced the amino terminus of the .beta..sub.2AR with T4 lysozyme (T4L). Several different amino-terminal fusion proteins were prepared and single particle EM was used to identify a fusion with a relatively fixed orientation of T4L in relation to the .beta..sub.2AR.

[0174] The second problem revealed by single particle EM analysis was increased variability in the positioning of the .alpha.-helical component of the G.alpha.s subunit. G.alpha.s consists of two domains, the ras-like GTPase domain (G.alpha.sRas), which interacts with the .beta..sub.2AR and the G.beta. subunit, and the .alpha.-helical domain (G.alpha.sAH). The interface of the two G.alpha.s subdomains forms the nucleotide-binding pocket (FIG. 7), and EM 2D averages and 3D reconstructions show that in the absence of guanine nucleotide, G.alpha.sAH has a variable position relative to the complex of T4L-.beta..sub.2AR-G.alpha.sRAS-G.beta..gamma. (FIG. 7b).

[0175] The variable position of G.alpha.sAH was attributed to the empty nucleotide-binding pocket. However, both GDP and nonhydrolyzable GTP analogs disrupt the .beta..sub.2AR-Gs complex (FIG. 13). The addition of pyrophosphate and its analog phosphonoformate (foscarnet) led to a significant increase in stabilization of G.alpha.sAH as determined by EM analysis of the detergent solubilized complex. Crystallization trials were carried out in Lipidic Cubic Phase (LCP) using a modified monolein designed to accommodate the large hydrophilic component of the T4L-.beta.2AR-Gs complex (Misquitta, L. V. et al. Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 2004 12, 2113-2124). Although we were able to obtain small crystals that diffracted to 7 .ANG., we were unable to improve their quality through the use of additives and other modifications.

[0176] In an effort to generate an antibody that would further stabilize the complex and facilitate crystallogenesis, .beta.2AR and the Gs heterotrimer were crosslinked with a small, homobifunctional amine-reactive crosslinker and used this stabilized complex to immunized llamas. Llamas and other camelids produce antibodies devoid of light chains. The single domain antigen binding fragments of these heavy chain only antibodies, known as nanobodies, are small (15 kDa), rigid and are easily cloned and expressed in E. coli. A nanobody (Nb35) was obtained that binds to the complex and prevents dissociation of the complex by GTP.gamma.S (FIG. 13). The T4L-.beta.2AR-Gs-Nb35 complex was used to obtain crystals that grew to 250 microns (FIG. 14) in LCP (monoolein 7.7) and diffracted to 2.9 .ANG.. A 3.2 .ANG. data set was obtained from 20 crystals and the structure was determined by molecular replacement.

[0177] The .beta..sub.2AR-Gs complex crystallized in space group P2.sub.1, with a single complex in each asymmetric unit. FIG. 8a shows the crystallographic packing interactions. Complexes are arrayed in alternating aqueous and lipidic layers with lattice contacts formed almost exclusively between soluble components of the complex, leaving receptor molecules suspended between G protein layers and widely separated from one another in the plane of the membrane. Extensive lattice contacts were formed among all the soluble proteins, likely accounting for the strong overall diffraction and remarkably clear electron density for the G protein. Nb35 and T4L facilitated crystal formation. Nb35 packs at the interface of G.beta. and G.alpha. subunits with complementarity determining region (CDR) 1 interacting primarily with G.beta. and a long CDR3 loop interacting with both G.beta. and G.alpha. subunits. The framework regions of Nb35 from one complex also interact with G.alpha. subunits from two adjacent complexes. T4L forms relatively sparse interactions with the amino terminus of the receptor, but packs against the amino terminus of the G.beta. subunit of one complex, the carboxyl terminus of the G.beta. subunit of another complex, and the G.beta. subunit of yet another complex. FIG. 8b shows the structure of the complete complex including T4L and Nb35, and FIG. 8c shows the .beta..sub.2AR-Gs complex alone.

Structure of the Active-State .beta.2AR

[0178] The .beta..sub.2AR-Gs structure provides the first high-resolution insight into the mechanism of signal transduction across the plasma membrane by a GPCR, and the structural basis for the functional properties of the ternary complex. FIG. 9a compares the structures of the agonist-bound receptor in the .beta..sub.2AR-Gs complex and the inactive carazolol-bound .beta..sub.2AR. The largest difference between the inactive and active structures is a 14 .ANG. outward movement of TM6 when measured at the C.alpha. carbon of E268. There is a smaller outward movement and extension of the cytoplasmic end of the TM5 helix by 7 residues. A stretch of 26 amino acids in the third intracellular loop (ICL3) is disordered. Another notable difference between inactive and active structures is the second intracellular loop (ICL2), which forms an extended loop in the inactive .beta..sub.2AR structure and an .alpha.-helix in the .beta..sub.2AR-Gs complex. This helix is also observed in the .beta..sub.2AR-Nb80 structure (FIG. 9b); however, it may not be a feature that is unique to the active state, since it is also observed in the inactive structure of the highly homologous avian .beta..sub.1AR.

[0179] The quality of the electron density maps for the .beta..sub.2AR is highest at this .beta..sub.2AR-G.alpha.sRas interface, and much weaker for the extracellular half, possibly due to the lack of crystal lattice contacts with the extracellular surface (FIG. 8a). As a result, we cannot confidently model the high-affinity agonist (BI-167107) in the ligand-binding pocket. However, the overall structure of the .beta..sub.2AR in the T4L-.beta..sub.2AR-Gs complex is very similar to our recent active-state structure of .beta..sub.2AR stabilized by a G protein mimetic nanobody (Nb80). These structures deviate primarily at the cytoplasmic ends of TMs 5 and 6 (FIG. 9b), possibly due to the presence of T4L that replaces ICL3 in the .beta..sub.2AR-Nb80 structure. Nonetheless, the .beta..sub.2AR-Nb80 complex exhibits the same high affinity for the agonist isoproterenol as does the .beta..sub.2AR-Gs complex, consistent with high structural homology around the ligand binding pocket. The electron density maps for the .beta..sub.2AR-Nb80 crystals provide a more reliable view of the conformational rearrangements of amino acids around the ligand-binding pocket and between the ligand-binding pocket and the Gs-coupling interface.

[0180] FIG. 9c shows the position of the highly conserved sequence motifs including D/ERY and NPxxY in the .beta..sub.2AR-Gs complex compared with the .beta..sub.2AR-Nb80 complex (see also Fig. S3). These conserved sequences have been proposed to be important for activation or for maintaining the receptor in the inactive state. The positions of these amino acids are essentially identical in these two structures demonstrating that Nb80 is a very good G protein surrogate. Only Arg131 differs between these two structures. In the .beta..sub.2AR-Nb80 structure Arg131 interacts with Nb80, whereas in the .beta..sub.2AR-Gs structure Arg131 packs against Tyr391 of G.alpha.s (FIG. 15).

[0181] The active state of the .beta..sub.2AR is stabilized by extensive interactions with (G.alpha.sRas) (FIG. 10). There are no direct interactions with G.beta. or G.gamma. subunits. The total buried surface of the .beta..sub.2AR-GsRas interface is 2576 .ANG..sup.2 (1300 .ANG..sup.2 for GsRas and 1276 .ANG..sup.2 for the .beta..sub.2AR). This interface is formed by ICL2, TM5 and TM6 of the .beta..sub.2AR, and by .alpha.5-helix, the .alpha.N-.beta.1 junction, the top of the .beta.3-strand, and the .alpha.4-helix of G.alpha.sRas (see Table 1 below for specific interactions). The .beta..sub.2AR sequences involved in this interaction have been shown to play a role in G protein coupling; however, there is no clear consensus sequence for Gs-coupling specificity when these segments are aligned with other GPCRs. Perhaps this is not surprising considering that the .beta..sub.2AR also couples to Gi and that many GPCRs couple to more than one G protein isoform. The structural basis for G protein coupling specificity must therefore involve more subtle features of the secondary and tertiary structure. Nevertheless, a noteworthy interaction involves Phe139, which is located at the beginning of the ICL2 helix and sits in a hydrophobic pocket formed by G.alpha.s His41 at the beginning of the .beta.1-strand, Val213 at the start of the .beta.3-strand and Phe376, Arg380 and Ile383 in the .alpha.5-helix (FIG. 4c). The .beta..sub.2AR mutant F139A displays severely impaired coupling to Gs. The residue corresponding to Phe139 is a Phe or Leu on almost all Gs coupled receptors, but is more variable in GPCRs known to couple to other G proteins. Of interest, the ICL2 helix is stabilized by an interaction between Asp130 of the conserved DRY sequence and Tyr141 in the middle of the ICL2 helix (FIG. 10c). Tyr141 has been shown to be a substrate for the insulin receptor tyrosine kinase; however, the functional significance of this phosphorylation is currently unknown.

Structure of Activated Gs

[0182] One surprising observation in the .beta..sub.2AR-Gs complex is the large displacement of the G.alpha.sAH relative to G.alpha.sRas (an approximately 127.degree. rotation about the junction between the domains) (FIG. 11a). In the crystal structure of G.alpha.s, the nucleotide-binding pocket is formed by the interface between G.alpha.sRas and G.alpha.sAH. Guanine nucleotide binding stabilizes the interaction between these two domains. The loss of this stabilizing effect of guanine nucleotide binding is consistent with the high flexibility observed for G.alpha.sAH in single particle EM analysis of the detergent solubilized complex. It is also in agreement with the increase in deuterium exchange at the interface between these two domains upon formation of the complex. Recently Hamm, Hubbell and colleagues, using double electron-electron resonance (DEER) spectroscopy, documented large (up to 20 .ANG.) changes in distance between nitroxide probes positioned on the Ras and .alpha.-helical domains of Gi upon formation of a complex with light-activated rhodopsin. Therefore, it is perhaps not surprising that GsAH is displaced relative to G.alpha.sRas; however, its location in this crystal structure most likely reflects only one of an ensemble of conformations that it can adopt under physiological conditions, but has been stabilized by crystal packing interactions.

[0183] The conformational links between the .beta..sub.2AR and the nucleotide-binding pocket primarily involve the amino and carboxyl terminal helices of G.alpha.s (FIG. 10). FIG. 11b focuses on the region of G.alpha.sRas that undergoes the largest conformational change when comparing the structure of G.alpha.sRas from the Gs-.beta..sub.2AR complex with that from the G.alpha.s-GTP.gamma.S complex. The largest difference is observed for the .alpha.5-helix, which is displaced 6 .ANG. towards the receptor and rotated as the carboxyl terminal end projects into transmembrane core of the .beta..sub.2AR. Associated with this movement, the .beta.6-.alpha.5 loop, which interacts with the guanine ring in the G.alpha.s-GTP.gamma.S structure, is displaced outward, away from the nucleotide-binding pocket (FIG. 11b-d). The movement of .alpha.5-helix is also associated with changes in interactions between this helix and the .beta.6-strand, the .alpha.N-.beta.1 loop, and the .alpha.1-helix. The .beta.1-strand forms another link between the .beta..sub.2AR and the nucleotide-binding pocket. The C-terminal end of this strand changes conformation around Gly47, and there are further changes in the .beta.1-.alpha.1 loop (P-loop) that coordinates the .gamma.-phosphate in the GTP-bound form (FIG. 11 b-d). The observations in the crystal structure are in agreement with deuterium exchange experiments where there is enhanced deuterium exchange in the .beta.1-strand and the amino terminal end of the .alpha.5-helix upon formation of the nucleotide-free .beta..sub.2AR-Gs complex. The DXMS studies provide additional insights into the dynamic nature of these conformational changes in Gs upon complex formation.

[0184] The structure of a GDP-bound Gs heterotrimer has not been determined in this study, so it is not possible to directly compare the G.alpha.s-G.beta..gamma. interface before and after formation of the .beta..sub.2AR-Gs complex. Based on the structure of the GDP-bound Gi heterotrimer, large changes in interactions between G.alpha.sRas and G.beta..gamma. upon formation of the complex with .beta..sub.2AR are not observed. This is also consistent with deuterium exchange studies. It should be noted that Nb35 binds at the interface between G.alpha.sRas and G.beta. (FIG. 8b). Therefore, we cannot exclude the possibility that Nb35 may influence the relative orientation of the G.alpha.sRas-G.beta..gamma. interface in the crystal structure. However, single particle EM studies provide evidence that Nb35 does not disrupt interactions between G.alpha.sAH and G.alpha.sRas.

Assembly of the .beta.2AR-Gs Complex

[0185] Clues to the initial stages of complex formation may come from the recent active state structures of rhodopsin. FIGS. 12a and b compare the active-state structure of .beta..sub.2AR in the .beta..sub.2AR-Gs complex with the recent structure of metarhodopsin II bound to the transducin peptide. The conformational changes in TM5 and TM6 are smaller in metarhodopsin II, and the position of the carboxyl terminal alpha helix of transducin is tilted by approximately 30.degree. relative to the position of the homologous region of Gs. These may represent fundamental differences in the receptor-G protein interactions between these two proteins, but given the strong conservation of the G-protein binding pocket, the changes more likely reflect the extensive contacts formed with the intact G protein. The position of the transducin peptide in metarhodopsin II may represent the initial interaction between a GDP-bound G protein and a GPCR. We have attempted to reproduce a similar complex between the .beta..sub.2AR and a synthetic peptide representing the carboxyl terminal 20 amino acids of Gs, but did not observe any effect of this peptide on receptor function, possibly due to the solubility and behavior of the peptide in solution. However, when the carboxyl terminal 20 amino acids of Gs are fused to the carboxyl terminus of the .beta..sub.2AR (FIG. 12c), we observe a 27-fold increase in agonist affinity (FIG. 12d). This effect is only 3.5-fold smaller than the effect we observe on agonist binding affinity in the .beta..sub.2AR-Gs complex, and demonstrates that there is a functional interaction between the peptide and receptor that may represent an initial stage in .beta..sub.2AR-Gs complex formation. FIG. 12 e, f presents a possible sequence of interactions of .beta..sub.2AR and Gs when forming the nucleotide free complex. The initial interaction of the .beta..sub.2AR with Gs would require an outward movement of the carboxyl terminus of the .alpha.5-helix away from the .beta.6-strand to permit interactions with the .beta..sub.2AR similar to those observed in metarhodopsin II. The dynamic character of the carboxyl terminal end of .alpha.5 is supported by deuterium exchange studies and the relatively loose packing of .alpha.5 with the rest of G.alpha.sRas in the structure of G.alpha.s alone. The subsequent formation of more extensive interactions between the .beta..sub.2AR ICL 2 and the amino terminus of G.alpha.s requires a rotation of G.alpha.sRas relative to the receptor and would be associated with further conformational changes in both .beta..sub.2AR and G.alpha.sRas (FIG. 12f). This binding model is in agreement with deuterium exchange experiments.

[0186] The coordinates and structure factors for the .beta..sub.2AR-Gs complex are deposited in the Protein Data Bank as accession number 3SN6, which is incorporated by reference herein.

TABLE-US-00001 TABLE 1 Potential intermolecular interaction within the R:G interface ##STR00001##

TABLE-US-00002 TABLE 2 Data collection and refinement statistics Data collection* Number of crystals 20 Space group P 2.sub.1 Cell dimensions a, b, c (.ANG.) 119.3, 64.6, 131.2 .alpha., .beta., .gamma. (.degree.) 90.0, 91.7, 90.0 Resolution (.ANG.) 41-3.2 (3.26-3.20) R.sub.merge (%) 15.6 (553) <I>/<.sigma.I> 10.8 (1.8) Completeness (%) 91.2 (53.9) Redundancy 6.5 (5.0) Refinement Resolution (.ANG.) 41-3.2 No. reflections 31075 (1557 in test set) R.sub.work/R.sub.free (%) 22.5/27.7 No. atoms 10277 No. protein residues 1318 Anisotropic B tensor B.sub.11 = -7.0/B.sub.22 = 4.7/B.sub.33 = 2.3/B.sub.13 = 2.1 Unmodelled sequences* .beta..sub.2 adrenergic receptor 29.sup.b, 176-178, 240-264, 342-365 G.sub.s.alpha., ras domain 1-8, 60-88, 203-204, 256-262 G.sub.s.gamma. 1-4, 63-68 T4 lysozyme 161.sup.c Average B-factors (.ANG..sup.2) .beta..sub.2 adrenergic receptor 133.5 G.sub.s.alpha., ras domain 82.8 G.sub.s.alpha., helical domain 123.0 G.sub.s.beta. 64.2 G.sub.s.gamma. 85.2 Nanobody 35 60.7 T4 lysozyme 113.7 R.m.s. deviation from ideality Bond length (.ANG.) 0.007 Bond angles (.degree.) 0.72 Ramachandran statistics.sup.d Favored regions (%) 95.8 Allowed regions (%) 4.2 Outliers (%) 0 *Highest shell statistics are in parentheses. .sup.aThese regions were omitted from the model due to poorly resolved electron density. Unmodelled purification tags are not included in these residue ranges. .sup.bResidues 1-28 of the .beta.2AR were omitted from the construct and T4L was fused to the amino terminus of transmembrane helix 1 to facilitate crystallization. .sup.cResidue 1 of T4L was omitted from the construct .sup.dAs defined by MolProbity.sup.3B.

TABLE-US-00003 TABLE 3 Data collection statistics by resolution shell Resolution Shell (.ANG.) <I>/<.sigma.I> R.sub.merge (%) Completeness (%) .sup. 41-8.67 18.8 06.6 97.1 8.67-6.89 16.9 09.2 99.5 6.89-6.02 14.4 13.0 99.7 6.02-5.47 12.8 16.7 99.9 5.47-5.08 13.4 15.9 99.9 5.08-4.78 13.4 16.9 99.8 4.78-4.54 12.2 18.2 99.6 4.54-4.34 11.6 20.1 99.8 4.34-4.18 9.5 22.9 99.4 4.18-4.03 7.7 26.2 99.1 4.03-3.91 6.6 27.9 98.7 3.91-3.79 5.3 30.2 98.7 3.79-3.69 3.8 36.6 96.7 3.69-3.60 4.6 36.9 94.6 3.60-3.52 2.3 45.7 90.3 3.52-3.45 2.2 47.9 86.3 3.45-3.38 2.4 45.6 80.5 3.38-3.31 2.1 47.3 69 3.31-3.26 2.2 49.8 59.4 3.26-3.20 1.8 55.3 53.9 Overall 10.8 15.6 91.2

Materials and Methods III

Generation of N-T4L Fused .beta.2AR Constructs

[0187] The human .beta..sub.2AR in the pFastbac1 Sf9 expression vector truncated at amino acid 365 in the cytoplasmic tail (.beta..sub.2AR365) was used as the starting template for generating the N-T4L fused .beta..sub.2AR constructs. The HA signal peptide followed by FLAG epitope tag and tobacco etch virus (TEV) protease recognition sequence were added to the N-terminus of the receptor to facilitate expression and purification. A point mutation of N187E was also introduced in the second extracellular loop to remove a glycosylation site (FIG. 22).

[0188] DNA cassettes encoding two different versions of T4L lysozyme (full length or with truncated C-terminus) with different numbers of additional alanines attached to the C-terminus were generated and amplified by PCR using the original .beta..sub.2AR-T4L .sup.3 as the template and synthetic oligonucleotides as primers. These different cassettes were inserted into the .beta..sub.2AR365 construct between the end of the TEV protease recognition sequence and Asp29, Glu30 or Val31 of the receptor as shown in (FIG. 22) by using the Quickchange multi protocol (Stratagene). Two point mutations M96T, M98T were also introduced into the .beta..sub.2AR sequence. Residues from Ser235 to Lys263 in the third intracellular loop were deleted with the Quickchange multi protocol using synthetic oligonucleotides as mutation primers. All the constructs were confirmed by DNA sequencing. The protein sequence of T4L-.beta..sub.2AR-.DELTA.-ICL3 is shown below:

TABLE-US-00004 (SEQ ID NO: 17) ##STR00002## DTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQ DVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLR MLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYAADEVWVV GMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGL AVVPFGAAHILTKTWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFA ITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINC YAEETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKROLOKID KFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLN WIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGE QSG.

[0189] The HA signal peptide is shown in italic letters; the FLAG epitope tag is shown in letters with underscore; the TEV recognition sequence is marked with a box and the cleavage site is shown with an asterisk; the full length T4L is shown in bold; the .beta..sub.2AR sequence from Asp29 to Gly365 excluding Ser235 to K263 is shown in bold underline, the 2-Ala linker is underlined).

[0190] The entire T4L-.beta..sub.2AR-.DELTA.-ICL3 gene described above was further cloned into the Best-Bac Sf9 expression vector pv11393 (expression systems) using the restriction enzyme digestion site XbaI and EcoRI. This version of T4L-.beta..sub.2AR-.DELTA.-ICL3 construct was also confirmed by DNA sequencing.

[0191] Whole Cell Binding to Assess the Expression Yield of Each Construct.

[0192] Recombinant baculovirus was made from the pFastbac1 Sf9 expression vector for each of the constructs illustrated in FIG. 22 using the Invitrogen protocol. Sf9 cells at a density of 4 million/ml were infected with second passage virus at different ratios of virus stock to cell culture (1:20, 1:50, and 1:100). After 48 hours, 5 .mu.l of the infected cells were incubated with 10 nM of [.sup.3H]-dihydroalprenolol (DHA) in 500 .mu.l of binding buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 5 mg/ml BSA). Cells were harvested and washed with cold binding buffer using a Brandel harvester. Bound [.sup.3H]DHA was measured with scintillation counter (Beckman). Non-specific binding of [.sup.3H]DHA was assessed by including 10 .mu.M of alprenolol (Sigma) in the same binding reaction. The expression level of each construct was determined using the specific activity of the bound [.sup.3H]DHA. Each experiment was performed in triplicate.

[0193] Saturation and Competition Binding Assays.

[0194] Membranes from Sf9 cells expressing either wild-type .beta.2AR or T4L-.beta..sub.2AR-.DELTA.-ICL3 were prepared based on a previously describe protocol.sup.12. In each reaction for the saturation binding assay, membranes containing approximately 0.2 pmol receptor were incubated with concentrations of [.sup.3H]DHA ranging from 5 pM to 10 nM in 500 .mu.l of buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 0.5 mg/ml BSA) at room temperature with shaking at 230 rpm for 1 hour. Membranes were isolated from free [.sup.3H]DHA using a Brandel harvester and washed three times with cold buffer. The amount of receptor bound [.sup.3H]DHA was measured using a scintillation counter (Beckman). Non-specific binding of the [.sup.3H]DHA in each reaction was assessed by including 1 .mu.M alprenolol (Sigma) in the same reaction. In each reaction for the competition binding assay, membrane containing approximately 0.2 pmol receptor was incubated with 1 nM [.sup.3H]DHA and different concentrations of (-)-isoproterenol (Sigma) ranging from 1 nM to 1 mM. Membranes were harvested and washed three times with cold buffer. The bound [.sup.3H]DHA was counted as described above. Non-specific [.sup.3H]DHA was assessed by replacing (-)-isoproterenol with 1 .mu.M alprenolol. All the binding data was analyzed by non-linear regression method using Graphpad Prism. Each experiment was performed in triplicate.

[0195] Expression and Purification of T4L-.beta..sub.2AR-.DELTA.-ICL3 from Baculovirus-Infected Sf9 Cells

[0196] Recombinant baculovirus was made from pv11393-T4L-.beta..sub.2AR-.DELTA.-ICL3 using Best-Bac expression system, as described by the system protocol (Expression Systems). T4L-.beta..sub.2AR-.DELTA.-ICL3 was expressed by infecting Sf9 cells at a density of 4 million/ml with a second passage baculovirus stock at a virus to cell ratio of 1:50. 1 .mu.M of the antagonist alprenolol was included to enhance the receptor stability and yield. The infected cells were harvested after 48 hs of incubation at 27.degree. C.

[0197] Cell pellets were lysed by vigorous stirring in lysis buffer (10 mM TRIS-Cl pH 7.5, 2 mM EDTA, 10 ml of buffer per gram of cell pellet) supplemented with protease inhibitor Leupeptin (2.5 .mu.g/ml final concentration, Sigma) and Benzamindine (160 .mu.g/ml final concentration, Sigma) for 15 minutes. The T4L-.beta..sub.2AR-.DELTA.-ICL3 protein was extracted from the cell membrane by dounce homogenization in solubilization buffer (100 mM NaCl, 20 mM TRIS-Cl, pH 7.5, 1% Dodecylmaltoside) supplemented with Leupeptin and Benzamindine (2.5 .mu.g/ml and 160 .mu.g/ml final concentration, respectively). 10 ml of solubilization buffer was used for each gram of cell pellet. The Dodecylmaltoside (DDM)-solubilized T4L-.beta..sub.2AR-.DELTA.-ICL3 bearing the FLAG epitope was then purified by M1 antibody affinity chromatography (Sigma). Extensive washing using HLS buffer (100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) was performed to get rid of alprenolol. The protein was then eluted with HLS buffer supplemented with 5 mM EDTA, 200 .mu.g/ml free FLAG peptide and a saturating concentration of cholesterol hemisuccinate.

[0198] The eluted T4L-.beta..sub.2AR-.DELTA.-ICL3 was further purified by affinity chromatography using alprenolol-Sepharose as previously described .sup.3 in order to isolate functional T4L-.beta..sub.2AR-.DELTA.-ICL3 from non-functional protein. HHS buffer (350 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) supplemented with 300 .mu.M alprenolol and a saturating concentration of cholesterol hemisuccinate was used to elute the protein. The eluted T4L-.beta..sub.2AR-.DELTA.-ICL3 bound with alprenolol was then re-applied to M1 resin, allowing exchanging alprenolol with carazolol in HHS buffer supplemented with 30 nM carazolol. T4L-.beta..sub.2AR-.DELTA.-ICL3 bound with carazolol was then eluted from M1 resin with HHS buffer supplemented with 5 mM EDTA, 200 .mu.g/ml free FLAG peptide and saturating concentration of cholesterol hemisuccinate. The FLAG epitope tag of T4L-.beta..sub.2AR-.DELTA.-ICL3 was removed by the treatment of tobacco etch virus (TEV) protease (invitrogen) for 3 hs at room temperature or overnight at 4.degree. C. The untagged T4L-.beta..sub.2AR-.DELTA.-ICL3-cazazolol complex was then further purified by chromatography (SEC) using S200 column (GE healthcare) equilibrated in 100 mM NaCl, 10 mM HEPES pH 7.5, 0.1% DDM and 1 nM carazolol. The same buffer was used as the running buffer for SEC. The purity of the final T4L-.beta..sub.2AR-.DELTA.ICL3 is more than 90% according to the result of SDS-PAGE electrophoresis.

[0199] Crystallization of the T4L-.beta..sub.2AR-.DELTA.ICL3-Carazolo Complex

[0200] The purified T4L-.beta..sub.2AR-.DELTA.-ICL3-carazolol complex was concentrated to a final concentration of 60 mg/ml using centricon Vivaspin (GE healthcare). The complex was crystallized using the lipid cubic phase (LCP) method as previously described.sup.3. The protein complex was mixed with lipid moloolein with a 1:1.5 mass ratio at room temperature. 0.030 of the protein-lipid mixture drop was deposited in each well of a 96-well glass sandwich plate (Molecular Dimensions). The drop was then overlaid with 0.65 .mu.l of precipitant and the well was sealed by glass coverslip. By using this method, the T4L-.beta..sub.2AR-.DELTA.-ICL3-carazolol complex was crystallized in 37% PEG300 (v/v), 0.1M Bis-Tris propane, pH 6.5, 0.1 M ammonium phosphate after 2 days of incubation in 20.degree. C.

[0201] Data Collection and Structure Determination

[0202] The crystals were harvested and frozen in liquid nitrogen directly without using additional cryo-protectant. Diffraction data from 15 different crystals was collected using the GM/CA-CAT minibeam at 23-ID-D, Advance Photon Source, Argonne National Labs. The data was processed with HKL2000 and the structure was solved by molecular replacement using Molrep. Further model rebuilding was performed by using coot and the structure was refined with Phenix. The validation of the final structural model was performed using Molprobity. Data processing and refinement statistics are shown in Table 4.

Results III

[0203] T4 lysozyme was fused to the N-terminus of the .beta..sub.2 adrenergic receptor (.beta..sub.2AR), a G-protein coupled receptor (GPCR) for catecholamines. The N-terminally fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

[0204] The N terminus of the .beta..sub.2AR was replaced with T4 lysozyme to produce a T4L-GPCR fusion. To have a T4L-.beta..sub.2AR construct suitable for crystallization, the link between T4L and the receptor should be relatively short and rigid, yet not interfere with receptor function. Several different constructs were generated and examined for expression levels and binding properties (FIG. 22). In an effort to generate a rigid interaction between T4L and the .beta..sub.2AR, we removed the relatively flexible C-terminus of the T4L and attempted to fuse the remaining C terminal helix of T4L with the extracellular end of TM1 of the .beta..sub.2AR. None of these constructs gave sufficient amounts of functional receptor.

[0205] In the second approach, we fused the carboxyl terminus of T4L to D29, the first amino acid of the extracellular helical extension of TM 1. Four constructs were generated and examined: direct fusion of T4L to D29, and the inclusion of 1-3 Ala residues between T4L and the .beta..sub.2AR (FIG. 22). The highest level of expression was obtained from the fusion with a 2-Ala linker. The fusion protein had normal pharmacology and G protein coupling. To improve expression, two additional point mutations M96T and M98T were made in the .beta..sub.2AR component of the fusion protein. We have previously observed that mutation of these residues, which are located in the first extracellular loop and face away from the protein, had no effect on receptor function, but enhanced expression by up to two-fold. We were able to produce 1.5 mg of pure, functional protein from 1 liter of Sf9 cells.

[0206] This version of T4L-.beta..sub.2AR was recently used to obtain the crystal structure of the .beta..sub.2AR-Gs complex. However, in this structure most of the lattice contacts in this crystal are mediated by Gs, and the N terminal fused T4L does not pack against the extracellular surface of its fused .beta..sub.2AR (FIG. 24). The lack of interactions between T4L and the extracellular surface of the .beta..sub.2AR in the .beta..sub.2AR-Gs complex suggested that T4L fused to the N terminus of the .beta..sub.2AR might not be sufficiently constrained to facilitate crystallogenesis in the absence of the cytoplasmic G protein. The amino terminal T4L facilitated crystallogenesis in the absence of a soluble protein bound or fused to the third intracellular loop. Additional modifications were made to minimize unstructured sequence in the third intracellular loop and carboxyl terminus (FIG. 22). The C-terminus was truncated after amino acid 365. The 3.sup.rd intracellular loop (ICL3) of .beta..sub.2AR is another flexible region and it is subject to proteolysis. This loop was truncated in the fusion protein by removing residues 235 to 263. The final construct T4L-.beta..sub.2AR-.DELTA.-ICL3 is illustrated in FIG. 22.

[0207] To determine the functional integrity of T4L-.beta..sub.2AR-.DELTA.-ICL3, agonist and antagonist binding affinities were determined. The ligand binding pocket is formed by amino acids from four transmembrane domains and is therefore very sensitive to any perturbation of the receptor structure. T4L-.beta..sub.2AR-.DELTA.-ICL3 exhibits ligand binding affinities for the antagonist [3H]-Dihydroalprenolol and the agonist isopreterenol that are comparable to those of the wild type receptor (FIG. 25).

[0208] Purified T4L-.beta..sub.2AR-.DELTA.-ICL3 bound to the inverse agonist carazolol crystallized as small rods in lipid cubic phase (37% PEG300 (v/v), 0.1M Bis-Tris propane, pH 6.5, 0.1 M ammonium phosphate). Crystals diffracted to a resolution of 3.3 .ANG.; however, due to radiation damage, our dataset was limited to 4.0 (Table 4). Nevertheless, the dataset allowed us to solve the structure by molecular replacement. The interaction between the .beta..sub.2AR and T4L is sufficiently rigid to detect electron density for the 2 Ala link between these two proteins (FIG. 26). This link was not detectable in the electron density map of the .beta..sub.2AR-Gs structure (FIG. 24). In the T4L-.beta..sub.2AR-.DELTA.-ICL3 crystal lattice, the packing interactions are primarily mediated by T4L and there are no contacts between adjacent receptors (FIG. 23), indicating the important role of the T4L in facilitating GPCR crystallization. Each T4L has four packing interactions: 1-against ECL1 and ECL2 of its fused .beta..sub.2AR-.DELTA.-ICL3, 2-against T4L of one adjacent T4L-.beta..sub.2AR-.DELTA.-ICL3, 3-against T4L, ECL2 and ECL3 of a second T4L-.beta..sub.2AR-.DELTA.-ICL3, and 4-against ICL3 and Helix 8 of a third T4L-.beta..sub.2AR-.DELTA.-ICL3 (FIG. 23).

[0209] The structures of the .beta..sub.2AR in T4L-.beta..sub.2AR-.DELTA.-ICL3 and .beta.2AR-T4L (pdb 2RH1) are very similar to each other (FIG. 27), with an overall root mean square deviation of 0.48 .ANG.. Only minor differences can be observed in these two structures, presumably due to different crystal packing patterns. The similarity of the structures determined independently through different strategies further validates the fusion protein approach, demonstrating that structural distortions due to protein engineering or crystal packing are unlikely.

[0210] Of interest, ICL2 in the two inactive structures of .beta..sub.2AR-Fab5 and .beta..sub.2AR-T4L is in an extended loop while it is an alpha helix in both active structures: the .beta..sub.2AR-Gs complex and the .beta..sub.2AR stabilized by Nb80. In both of the inactive structures (.beta..sub.2AR-Fab5 and .beta..sub.2AR-T4L), ICL2 participates in lattice contacts that may influence its conformation. However, in the T4L-.beta.2AR-.DELTA.-ICL3 structure ICL2 is not involved in packing interactions, yet is an extended loop is nearly identical to that observed in the other inactive state .beta..sub.2AR structures (FIG. 27). Thus, this extended loop structure may reflect an inactive state.

[0211] In conclusion, fusion of T4L to the amino terminus of a GPCR can facilitate crystallogenesis. This approach can also facilitate the formation of crystals of a GPCR in complex with a cytoplasmic signaling protein.

[0212] FIG. 28 illustrates shows the structure of T4L-.beta.2AR fusion bound to salmeterol, a partial agonist used to treat asthma. In this structure, the partial-active state is stabilized by a nanobody (nanobody 71). This structure was obtained using similar methods to those described above.

TABLE-US-00005 TABLE 4 Data collection Space group P2.sub.12.sub.12.sub.1 Unit cell dimensions a, b, c (.ANG.) 51.4, 71.4, 161.4 Resolution (.ANG.) 50-4.0 (4.07-4.00)* R.sub.merge 0.199 (0.799) <I/.sigma.I> 8.4 (1.5) Completeness (%) 84.3 (71.2) Multiplicity 4.7 (3.7) Refinement Resolution (.ANG.) 30-3.99 No. reflections work/free 4547/691 R.sub.work/R.sub.free 0.267/0.293 No. atoms 3623 Average B values (.ANG..sub.2) Receptor 197 T4L 177 Carazolol 160 Overall anisotropic B (.ANG..sub.2) B11/B22/B33 -21.2/59.3/-38.0 R.m.s deviations Bond lengths (.ANG.) 0.004 Bond angles (.degree.) 0.6764 Ramachandran plot** % favored 96.4 allowed 3.6 generously allowed 0.0 disallowed 0.0 *High resolution shell in parenthesis. **As defined by Molprobity R.sub.merge = .SIGMA..sub.hkI .SIGMA..sub.i|I.sub.i - <I>/.SIGMA..sub.hki.SIGMA..sub.iI.sub.i

Sequence CWU 1

1

291530PRTArtificial Sequencesynthetic fusion protein 1Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Asp Glu Val Trp Val Val Gly Met Gly Ile Val Met Ser Leu Ile 195 200 205 Val Leu Ala Ile Val Phe Gly Asn Val Leu Val Ile Thr Ala Ile Ala 210 215 220 Lys Phe Glu Arg Leu Gln Thr Val Thr Asn Tyr Phe Ile Thr Ser Leu 225 230 235 240 Ala Cys Ala Asp Leu Val Met Gly Leu Ala Val Val Pro Phe Gly Ala 245 250 255 Ala His Ile Leu Thr Lys Thr Trp Thr Phe Gly Asn Phe Trp Cys Glu 260 265 270 Phe Trp Thr Ser Ile Asp Val Leu Cys Val Thr Ala Ser Ile Glu Thr 275 280 285 Leu Cys Val Ile Ala Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe 290 295 300 Lys Tyr Gln Ser Leu Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu 305 310 315 320 Met Val Trp Ile Val Ser Gly Leu Thr Ser Phe Leu Pro Ile Gln Met 325 330 335 His Trp Tyr Arg Ala Thr His Gln Glu Ala Ile Asn Cys Tyr Ala Glu 340 345 350 Glu Thr Cys Cys Asp Phe Phe Thr Asn Gln Ala Tyr Ala Ile Ala Ser 355 360 365 Ser Ile Val Ser Phe Tyr Val Pro Leu Val Ile Met Val Phe Val Tyr 370 375 380 Ser Arg Val Phe Gln Glu Ala Lys Arg Gln Leu Gln Lys Ile Asp Lys 385 390 395 400 Ser Glu Gly Arg Phe His Val Gln Asn Leu Ser Gln Val Glu Gln Asp 405 410 415 Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Leu Lys 420 425 430 Glu His Lys Ala Leu Lys Thr Leu Gly Ile Ile Met Gly Thr Phe Thr 435 440 445 Leu Cys Trp Leu Pro Phe Phe Ile Val Asn Ile Val His Val Ile Gln 450 455 460 Asp Asn Leu Ile Arg Lys Glu Val Tyr Ile Leu Leu Asn Trp Ile Gly 465 470 475 480 Tyr Val Asn Ser Gly Phe Asn Pro Leu Ile Tyr Cys Arg Ser Pro Asp 485 490 495 Phe Arg Ile Ala Phe Gln Glu Leu Leu Cys Leu Arg Arg Ser Ser Leu 500 505 510 Lys Ala Tyr Gly Asn Gly Tyr Ser Ser Asn Gly Asn Thr Gly Glu Gln 515 520 525 Ser Gly 530 258PRTBos taurus 2Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45 Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 376PRTBos taurus 3Met Lys Ser Pro Glu Glu Leu Lys Gly Ile Phe Glu Lys Tyr Ala Ala 1 5 10 15 Lys Glu Gly Asp Pro Asn Gln Leu Ser Lys Glu Glu Leu Lys Leu Leu 20 25 30 Leu Gln Thr Glu Phe Pro Ser Leu Leu Lys Gly Pro Ser Thr Leu Asp 35 40 45 Glu Leu Phe Glu Glu Leu Asp Lys Asn Gly Asp Gly Glu Val Ser Phe 50 55 60 Glu Glu Phe Gln Val Leu Val Lys Lys Ile Ser Gln 65 70 75 4111PRTBacillus amyloliquefaciens 4Met Ala Gln Val Ile Asn Thr Phe Asp Gly Val Ala Asp Tyr Leu Gln 1 5 10 15 Thr Tyr His Lys Leu Pro Asp Asn Tyr Ile Thr Lys Ser Glu Ala Gln 20 25 30 Ala Leu Gly Trp Val Ala Ser Lys Gly Asn Leu Ala Asp Val Ala Pro 35 40 45 Gly Lys Ser Ile Gly Gly Asp Ile Phe Ser Asn Arg Glu Gly Lys Leu 50 55 60 Pro Gly Lys Ser Gly Arg Thr Trp Arg Glu Ala Asp Ile Asn Tyr Thr 65 70 75 80 Ser Gly Phe Arg Asn Ser Asp Arg Ile Leu Tyr Ser Ser Asp Trp Leu 85 90 95 Ile Tyr Lys Thr Thr Asp His Tyr Gln Thr Phe Thr Lys Ile Arg 100 105 110 5190PRTTrichoderma reesei 5Glu Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser 1 5 10 15 Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Gly 20 25 30 Gly Gln Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly 35 40 45 Lys Gly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly 50 55 60 Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser 65 70 75 80 Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr 85 90 95 Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly 100 105 110 Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile 115 120 125 Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His 130 135 140 Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala 145 150 155 160 Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val 165 170 175 Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser 180 185 190 6455PRTPyrococcus furiosus 6Met Pro Thr Trp Glu Glu Leu Tyr Lys Asn Ala Ile Glu Lys Ala Ile 1 5 10 15 Lys Ser Val Pro Lys Val Lys Gly Val Leu Leu Gly Tyr Asn Thr Asn 20 25 30 Ile Asp Ala Ile Lys Tyr Leu Asp Ser Lys Asp Leu Glu Glu Arg Ile 35 40 45 Ile Lys Ala Gly Lys Glu Glu Val Ile Lys Tyr Ser Glu Glu Leu Pro 50 55 60 Asp Lys Ile Asn Thr Val Ser Gln Leu Leu Gly Ser Ile Leu Trp Ser 65 70 75 80 Ile Arg Arg Gly Lys Ala Ala Glu Leu Phe Val Glu Ser Cys Pro Val 85 90 95 Arg Phe Tyr Met Lys Arg Trp Gly Trp Asn Glu Leu Arg Met Gly Gly 100 105 110 Gln Ala Gly Ile Met Ala Asn Leu Leu Gly Gly Val Tyr Gly Val Pro 115 120 125 Val Ile Val His Val Pro Gln Leu Ser Arg Leu Gln Ala Asn Leu Phe 130 135 140 Leu Asp Gly Pro Ile Tyr Val Pro Thr Leu Glu Asn Gly Glu Val Lys 145 150 155 160 Leu Ile His Pro Lys Glu Phe Ser Gly Asp Glu Glu Asn Cys Ile His 165 170 175 Tyr Ile Tyr Glu Phe Pro Arg Gly Phe Arg Val Phe Glu Phe Glu Ala 180 185 190 Pro Arg Glu Asn Arg Phe Ile Gly Ser Ala Asp Asp Tyr Asn Thr Thr 195 200 205 Leu Phe Ile Arg Glu Glu Phe Arg Glu Ser Phe Ser Glu Val Ile Lys 210 215 220 Asn Val Gln Leu Ala Ile Leu Ser Gly Leu Gln Ala Leu Thr Lys Glu 225 230 235 240 Asn Tyr Lys Glu Pro Phe Glu Ile Val Lys Ser Asn Leu Glu Val Leu 245 250 255 Asn Glu Arg Glu Ile Pro Val His Leu Glu Phe Ala Phe Thr Pro Asp 260 265 270 Glu Lys Val Arg Glu Glu Ile Leu Asn Val Leu Gly Met Phe Tyr Ser 275 280 285 Val Gly Leu Asn Glu Val Glu Leu Ala Ser Ile Met Glu Ile Leu Gly 290 295 300 Glu Lys Lys Leu Ala Lys Glu Leu Leu Ala His Asp Pro Val Asp Pro 305 310 315 320 Ile Ala Val Thr Glu Ala Met Leu Lys Leu Ala Lys Lys Thr Gly Val 325 330 335 Lys Arg Ile His Phe His Thr Tyr Gly Tyr Tyr Leu Ala Leu Thr Glu 340 345 350 Tyr Lys Gly Glu His Val Arg Asp Ala Leu Leu Phe Ala Ala Leu Ala 355 360 365 Ala Ala Ala Lys Ala Met Lys Gly Asn Ile Thr Ser Leu Glu Glu Ile 370 375 380 Arg Glu Ala Thr Ser Val Pro Val Asn Glu Lys Ala Thr Gln Val Glu 385 390 395 400 Glu Lys Leu Arg Ala Glu Tyr Gly Ile Lys Glu Gly Ile Gly Glu Val 405 410 415 Glu Gly Tyr Gln Ile Ala Phe Ile Pro Thr Lys Ile Val Ala Lys Pro 420 425 430 Lys Ser Thr Val Gly Ile Gly Asp Thr Ile Ser Ser Ser Ala Phe Ile 435 440 445 Gly Glu Phe Ser Phe Thr Leu 450 455 7576PRTArtificial Sequencesynthetic fusion protein 7Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Thr Ala Cys Lys Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile 195 200 205 Leu Ile Thr Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu 210 215 220 Asn Arg Arg Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala 225 230 235 240 Ile Thr Asp Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile 245 250 255 Tyr Gln Leu Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile 260 265 270 Tyr Thr Ser Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu 275 280 285 Phe Met Ile Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg 290 295 300 Tyr Pro Val Leu Val Thr Pro Val Arg Val Ala Ile Ser Leu Val Leu 305 310 315 320 Ile Trp Val Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly 325 330 335 Trp Asn Ser Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys 340 345 350 Cys Lys Val Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val 355 360 365 Thr Phe Tyr Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile 370 375 380 Phe Lys Val Ala Arg Asp Gln Ala Lys Arg Ile Asn His Ile Ser Ser 385 390 395 400 Trp Lys Ala Ala Thr Ile Arg Glu His Lys Ala Thr Val Thr Leu Ala 405 410 415 Ala Val Met Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala 420 425 430 Phe Val Tyr Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Glu Val Leu 435 440 445 Glu Ala Ile Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro 450 455 460 Ile Leu Tyr Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln 465 470 475 480 Leu Phe Cys Cys Arg Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu 485 490 495 Arg Ser Asn Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg 500 505 510 Gln Gln Glu Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu 515 520 525 Val Thr Ala Pro Gln Gly Ala Thr Asp Arg Pro Trp Leu Cys Leu Pro 530 535 540 Glu Cys Trp Ser Val Glu Leu Thr His Ser Phe Ile His Leu Phe Ile 545 550 555 560 His Ser Phe Ala Asn Ile His Pro Ile Pro Thr Thr Cys Gln Glu Leu 565 570 575 8594PRTArtificial Sequencesynthetic fusion protein 8Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu Thr Ala Val Val Ile 195 200 205 Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile Met Ala Val Ser Leu 210 215 220 Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala 225 230 235 240 Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met Pro Val Ser Met Leu 245 250 255 Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro Ser Lys Leu Cys Ala 260 265 270 Val Trp Ile Tyr Leu Asp Val Leu

Phe Ser Thr Ala Ser Ile Met His 275 280 285 Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala Ile Gln Asn Pro Ile 290 295 300 His His Ser Arg Phe Asn Ser Arg Thr Lys Ala Phe Leu Lys Ile Ile 305 310 315 320 Ala Val Trp Thr Ile Ser Val Gly Ile Ser Met Pro Ile Pro Val Phe 325 330 335 Gly Leu Gln Asp Asp Ser Lys Val Phe Lys Glu Gly Ser Cys Leu Leu 340 345 350 Ala Asp Asp Asn Phe Val Leu Ile Gly Ser Phe Val Ser Phe Phe Ile 355 360 365 Pro Leu Thr Ile Met Val Ile Thr Tyr Phe Leu Thr Ile Lys Ser Leu 370 375 380 Gln Lys Glu Ala Thr Leu Cys Val Ser Asp Leu Gly Thr Arg Ala Lys 385 390 395 400 Leu Ala Ser Phe Ser Phe Leu Pro Gln Ser Ser Leu Ser Ser Glu Lys 405 410 415 Leu Phe Gln Arg Ser Ile His Arg Glu Pro Gly Ser Tyr Thr Gly Arg 420 425 430 Arg Thr Met Gln Ser Ile Ser Asn Glu Gln Lys Ala Cys Lys Val Leu 435 440 445 Gly Ile Val Phe Phe Leu Phe Val Val Met Trp Cys Pro Phe Phe Ile 450 455 460 Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser Cys Asn Glu Asp Val 465 470 475 480 Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile Gly Tyr Leu Ser Ser 485 490 495 Ala Val Asn Pro Leu Val Tyr Thr Leu Phe Asn Lys Thr Tyr Arg Ser 500 505 510 Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys Glu Asn Lys Lys Pro 515 520 525 Leu Gln Leu Ile Leu Val Asn Thr Ile Pro Ala Leu Ala Tyr Lys Ser 530 535 540 Ser Gln Leu Gln Met Gly Gln Lys Lys Asn Ser Lys Gln Asp Ala Lys 545 550 555 560 Thr Thr Asp Asn Asp Cys Ser Met Val Ala Leu Gly Lys Gln His Ser 565 570 575 Glu Glu Ala Ser Lys Asp Asn Ser Asp Gly Val Asn Glu Lys Val Ser 580 585 590 Cys Val 9530PRTArtificial Sequencesynthetic fusion protein 9Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Arg His Asn Tyr Ile Phe Val Met Ile Pro Thr Leu Tyr Ser Ile 195 200 205 Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu Val Val Ile Val Ile 210 215 220 Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser Val Phe Leu Leu Asn 225 230 235 240 Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr Leu Pro Leu Trp Ala 245 250 255 Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe Gly Asn Tyr Leu Cys 260 265 270 Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu Tyr Ala Ser Val Phe 275 280 285 Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu Ala Ile Val His Pro 290 295 300 Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val Ala Lys Val Thr Cys 305 310 315 320 Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser Leu Pro Ala Ile Ile 325 330 335 His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn Ile Thr Val Cys Ala 340 345 350 Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro Ile Gly Leu Gly Leu 355 360 365 Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe Leu Ile Ile Leu Thr 370 375 380 Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys Ala Tyr Glu Ile Gln 385 390 395 400 Lys Asn Lys Pro Arg Asn Asp Asp Ile Phe Lys Ile Ile Met Ala Ile 405 410 415 Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His Gln Ile Phe Thr Phe 420 425 430 Leu Asp Val Leu Ile Gln Leu Gly Ile Ile Arg Asp Cys Arg Ile Ala 435 440 445 Asp Ile Val Asp Thr Ala Met Pro Ile Thr Ile Cys Ile Ala Tyr Phe 450 455 460 Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe Leu Gly Lys Lys Phe 465 470 475 480 Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile Pro Pro Lys Ala Lys 485 490 495 Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr Leu Ser Tyr Arg Pro 500 505 510 Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro Ala Pro Cys Phe Glu 515 520 525 Val Glu 530 10528PRTArtificial Sequencesynthetic fusion protein 10Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Ser Met Ile Thr Ala Thr Thr Ile Met Ala Leu Tyr Ser Ile Val 195 200 205 Cys Val Val Gly Leu Phe Gly Asn Phe Leu Val Met Tyr Val Ile Val 210 215 220 Arg Tyr Thr Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu 225 230 235 240 Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val 245 250 255 Asn Tyr Leu Met Gly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile 260 265 270 Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu 275 280 285 Cys Thr Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys 290 295 300 Ala Leu Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Ile Asn Val Cys 305 310 315 320 Asn Trp Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met Ala 325 330 335 Thr Thr Lys Tyr Arg Gln Gly Ser Ile Asp Cys Thr Leu Thr Phe Ser 340 345 350 His Pro Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys Val Phe Ile 355 360 365 Phe Ala Phe Ile Met Pro Val Leu Ile Ile Thr Val Cys Tyr Gly Leu 370 375 380 Met Ile Leu Arg Leu Lys Ser Val Arg Met Leu Ser Gly Ser Lys Glu 385 390 395 400 Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg Met Val Leu Val Val Val 405 410 415 Ala Val Phe Ile Val Cys Trp Thr Pro Ile His Ile Tyr Val Ile Ile 420 425 430 Lys Ala Leu Val Thr Ile Pro Glu Thr Thr Phe Gln Thr Val Ser Trp 435 440 445 His Phe Cys Ile Ala Leu Gly Tyr Thr Asn Ser Cys Leu Asn Pro Val 450 455 460 Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Glu Phe 465 470 475 480 Cys Ile Pro Thr Ser Ser Asn Ile Glu Gln Gln Asn Ser Thr Arg Ile 485 490 495 Arg Gln Asn Thr Arg Asp His Pro Ser Thr Ala Asn Thr Val Asp Arg 500 505 510 Thr Asn His Gln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro 515 520 525 11543PRTArtificial Sequencesynthetic fusion protein 11Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Leu Pro Leu Ala Met Ile Phe Thr Leu Ala Leu Ala Tyr Gly Ala 195 200 205 Val Ile Ile Leu Gly Val Ser Gly Asn Leu Ala Leu Ile Ile Ile Ile 210 215 220 Leu Lys Gln Lys Glu Met Arg Asn Val Thr Asn Ile Leu Ile Val Asn 225 230 235 240 Leu Ser Phe Ser Asp Leu Leu Val Ala Ile Met Cys Leu Pro Leu Thr 245 250 255 Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly Glu Ala Met Cys 260 265 270 Lys Leu Asn Pro Phe Val Gln Cys Val Ser Ile Thr Val Ser Ile Phe 275 280 285 Ser Leu Val Leu Ile Ala Val Glu Arg His Gln Leu Ile Ile Asn Pro 290 295 300 Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr Val Gly Ile Ala Val 305 310 315 320 Ile Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe Leu Ile Tyr Gln 325 330 335 Val Met Thr Asp Glu Pro Phe Gln Asn Val Thr Leu Asp Ala Tyr Lys 340 345 350 Asp Lys Tyr Val Cys Phe Asp Gln Phe Pro Ser Asp Ser His Arg Leu 355 360 365 Ser Tyr Thr Thr Leu Leu Leu Val Leu Gln Tyr Phe Gly Pro Leu Cys 370 375 380 Phe Ile Phe Ile Cys Tyr Phe Lys Ile Tyr Ile Arg Leu Lys Arg Arg 385 390 395 400 Asn Asn Met Met Asp Lys Met Arg Asp Asn Lys Tyr Arg Ser Ser Glu 405 410 415 Thr Lys Arg Ile Asn Ile Met Leu Leu Ser Ile Val Val Ala Phe Ala 420 425 430 Val Cys Trp Leu Pro Leu Thr Ile Phe Asn Thr Val Phe Asp Trp Asn 435 440 445 His Gln Ile Ile Ala Thr Cys Asn His Asn Leu Leu Phe Leu Leu Cys 450 455 460 His Leu Thr Ala Met Ile Ser Thr Cys Val Asn Pro Ile Phe Tyr Gly 465 470 475 480 Phe Leu Asn Lys Asn Phe Gln Arg Asp Leu Gln Phe Phe Phe Asn Phe 485 490 495 Cys Asp Phe Arg Ser Arg Asp Asp Asp Tyr Glu Thr Ile Ala Met Ser 500 505 510 Thr Met His Thr Asp Val Ser Lys Thr Ser Leu Lys Gln Ala Ser Pro 515 520 525 Val Ala Phe Lys Lys Ile Asn Asn Asn Asp Asp Asn Glu Lys Ile 530 535 540 12482PRTArtificial Sequencesynthetic fusion protein 12Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Trp Pro His Leu Glu Val Val Ile Phe Val Val Val Leu Ile Phe 195 200 205 Tyr Leu Met Thr Leu Ile Gly Asn Leu Phe Ile Ile Ile Leu Ser Tyr 210 215 220 Leu Asp Ser His Leu His Thr Pro Met Tyr Phe Phe Leu Ser Asn Leu 225 230 235 240 Ser Phe Leu Asp Leu Cys Tyr Thr Thr Ser Ser Ile Pro Gln Leu Leu 245 250 255 Val Asn Leu Trp Gly Pro Glu Lys Thr Ile Ser Tyr Ala Gly Cys Met 260 265 270 Ile Gln Leu Tyr Phe Val Leu Ala Leu Gly Thr Ala Glu Cys Val Leu 275 280 285 Leu Val Val Met Ser Tyr Asp Arg Tyr Ala Ala Val Cys Arg Pro Leu 290 295 300 His Tyr Thr Val Leu Met His Pro Arg Phe Cys His Leu Leu Ala Val 305 310 315 320 Ala Ser Trp Val Ser Gly Phe Thr Asn Ser Ala Leu His Ser Ser Phe 325 330 335 Thr Phe Trp Val Pro Leu Cys Gly His Arg Gln Val Asp His Phe Phe 340 345 350 Cys Glu Val Pro Ala Leu Leu Arg Leu Ser Cys Val Asp Thr His Val 355

360 365 Asn Glu Leu Thr Leu Met Ile Thr Ser Ser Ile Phe Val Leu Ile Pro 370 375 380 Leu Ile Leu Ile Leu Thr Ser Tyr Gly Ala Ile Val Gln Ala Val Leu 385 390 395 400 Arg Met Gln Ser Thr Thr Gly Leu Gln Lys Val Phe Gly Thr Cys Gly 405 410 415 Ala His Leu Met Ala Val Ser Leu Phe Phe Ile Pro Ala Met Cys Ile 420 425 430 Tyr Leu Gln Pro Pro Ser Gly Asn Ser Gln Asp Gln Gly Lys Phe Ile 435 440 445 Ala Leu Phe Tyr Thr Val Val Thr Pro Ser Leu Asn Pro Leu Ile Tyr 450 455 460 Thr Leu Arg Asn Lys Val Val Arg Gly Ala Val Lys Arg Leu Met Gly 465 470 475 480 Trp Glu 13570PRTArtificial Sequencesynthetic fusion protein 13Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Leu Glu Tyr Gln Val Val Thr Ile Leu Leu Val Leu Ile Ile Cys 195 200 205 Gly Leu Gly Ile Val Gly Asn Ile Met Val Val Leu Val Val Met Arg 210 215 220 Thr Lys His Met Arg Thr Pro Thr Asn Cys Tyr Leu Val Ser Leu Ala 225 230 235 240 Val Ala Asp Leu Met Val Leu Val Ala Ala Gly Leu Pro Asn Ile Thr 245 250 255 Asp Ser Ile Tyr Gly Ser Trp Val Tyr Gly Tyr Val Gly Cys Leu Cys 260 265 270 Ile Thr Tyr Leu Gln Tyr Leu Gly Ile Asn Ala Ser Ser Cys Ser Ile 275 280 285 Thr Ala Phe Thr Ile Glu Arg Tyr Ile Ala Ile Cys His Pro Ile Lys 290 295 300 Ala Gln Phe Leu Cys Thr Phe Ser Arg Ala Lys Lys Ile Ile Ile Phe 305 310 315 320 Val Trp Ala Phe Thr Ser Leu Tyr Cys Met Leu Trp Phe Phe Leu Leu 325 330 335 Asp Leu Asn Ile Ser Thr Tyr Lys Asp Ala Ile Val Ile Ser Cys Gly 340 345 350 Tyr Lys Ile Ser Arg Asn Tyr Tyr Ser Pro Ile Tyr Leu Met Asp Phe 355 360 365 Gly Val Phe Tyr Val Val Pro Met Ile Leu Ala Thr Val Leu Tyr Gly 370 375 380 Phe Ile Ala Arg Ile Leu Phe Leu Asn Pro Ile Pro Ser Asp Pro Lys 385 390 395 400 Glu Asn Ser Lys Thr Trp Lys Asn Asp Ser Thr His Gln Asn Thr Asn 405 410 415 Leu Asn Val Asn Thr Ser Asn Arg Cys Phe Asn Ser Thr Val Ser Ser 420 425 430 Arg Lys Gln Val Thr Lys Met Leu Ala Val Val Val Ile Leu Phe Ala 435 440 445 Leu Leu Trp Met Pro Tyr Arg Thr Leu Val Val Val Asn Ser Phe Leu 450 455 460 Ser Ser Pro Phe Gln Glu Asn Trp Phe Leu Leu Phe Cys Arg Ile Cys 465 470 475 480 Ile Tyr Leu Asn Ser Ala Ile Asn Pro Val Ile Tyr Asn Leu Met Ser 485 490 495 Gln Lys Phe Arg Ala Ala Phe Arg Lys Leu Cys Asn Cys Lys Gln Lys 500 505 510 Pro Thr Glu Lys Pro Ala Asn Tyr Ser Val Ala Leu Asn Tyr Ser Val 515 520 525 Ile Lys Glu Ser Asp His Phe Ser Thr Glu Leu Asp Asp Ile Thr Val 530 535 540 Thr Asp Thr Tyr Leu Ser Ala Thr Lys Val Ser Phe Asp Asp Thr Cys 545 550 555 560 Leu Ala Ser Glu Val Ser Phe Ser Gln Ser 565 570 148PRTArtificial Sequencesynthetic peptide 14Asp Tyr Lys Asp Asp Asp Asp Ala 1 5 157PRTArtificial Sequencesynthetic peptide 15Glu Asn Leu Tyr Phe Gln Gly 1 5 169PRTArtificial Sequencesynthetic peptide 16Ser Glu Asn Leu Tyr Phe Gln Gly Ser 1 5 17502PRTArtificial Sequencesynthetic fusion protein 17Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly Asn 20 25 30 Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr 35 40 45 Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr 50 55 60 Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile 65 70 75 80 Gly Arg Asn Thr Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu 85 90 95 Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala 100 105 110 Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala 115 120 125 Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 130 135 140 Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala 145 150 155 160 Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala 165 170 175 Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala 180 185 190 Ala Asp Glu Val Trp Val Val Gly Met Gly Ile Val Met Ser Leu Ile 195 200 205 Val Leu Ala Ile Val Phe Gly Asn Val Leu Val Ile Thr Ala Ile Ala 210 215 220 Lys Phe Glu Arg Leu Gln Thr Val Thr Asn Tyr Phe Ile Thr Ser Leu 225 230 235 240 Ala Cys Ala Asp Leu Val Met Gly Leu Ala Val Val Pro Phe Gly Ala 245 250 255 Ala His Ile Leu Thr Lys Thr Trp Thr Phe Gly Asn Phe Trp Cys Glu 260 265 270 Phe Trp Thr Ser Ile Asp Val Leu Cys Val Thr Ala Ser Ile Glu Thr 275 280 285 Leu Cys Val Ile Ala Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe 290 295 300 Lys Tyr Gln Ser Leu Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu 305 310 315 320 Met Val Trp Ile Val Ser Gly Leu Thr Ser Phe Leu Pro Ile Gln Met 325 330 335 His Trp Tyr Arg Ala Thr His Gln Glu Ala Ile Asn Cys Tyr Ala Glu 340 345 350 Glu Thr Cys Cys Asp Phe Phe Thr Asn Gln Ala Tyr Ala Ile Ala Ser 355 360 365 Ser Ile Val Ser Phe Tyr Val Pro Leu Val Ile Met Val Phe Val Tyr 370 375 380 Ser Arg Val Phe Gln Glu Ala Lys Arg Gln Leu Gln Lys Ile Asp Lys 385 390 395 400 Phe Cys Leu Lys Glu His Lys Ala Leu Lys Thr Leu Gly Ile Ile Met 405 410 415 Gly Thr Phe Thr Leu Cys Trp Leu Pro Phe Phe Ile Val Asn Ile Val 420 425 430 His Val Ile Gln Asp Asn Leu Ile Arg Lys Glu Val Tyr Ile Leu Leu 435 440 445 Asn Trp Ile Gly Tyr Val Asn Ser Gly Phe Asn Pro Leu Ile Tyr Cys 450 455 460 Arg Ser Pro Asp Phe Arg Ile Ala Phe Gln Glu Leu Leu Cys Leu Arg 465 470 475 480 Arg Ser Ser Leu Lys Ala Tyr Gly Asn Gly Tyr Ser Ser Asn Gly Asn 485 490 495 Thr Gly Glu Gln Ser Gly 500 1831PRTArtificial Sequencesynthetic peptide 18Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Leu Tyr Phe Gln Gly 20 25 30 19413PRTArtificial Sequencesynthetic fusion protein 19Met Gly Gln Pro Gly Asn Gly Ser Ala Phe Leu Leu Ala Pro Asn Arg 1 5 10 15 Ser His Ala Pro Asp His Asp Val Thr Gln Gln Arg Asp Glu Val Trp 20 25 30 Val Val Gly Met Gly Ile Val Met Ser Leu Ile Val Leu Ala Ile Val 35 40 45 Phe Gly Asn Val Leu Val Ile Thr Ala Ile Ala Lys Phe Glu Arg Leu 50 55 60 Gln Thr Val Thr Asn Tyr Phe Ile Thr Ser Leu Ala Cys Ala Asp Leu 65 70 75 80 Val Met Gly Leu Ala Val Val Pro Phe Gly Ala Ala His Ile Leu Thr 85 90 95 Lys Thr Trp Thr Phe Gly Asn Phe Trp Cys Glu Phe Trp Thr Ser Ile 100 105 110 Asp Val Leu Cys Val Thr Ala Ser Ile Glu Thr Leu Cys Val Ile Ala 115 120 125 Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe Lys Tyr Gln Ser Leu 130 135 140 Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu Met Val Trp Ile Val 145 150 155 160 Ser Gly Leu Thr Ser Phe Leu Pro Ile Gln Met His Trp Tyr Arg Ala 165 170 175 Thr His Gln Glu Ala Ile Asn Cys Tyr Ala Glu Glu Thr Cys Cys Asp 180 185 190 Phe Phe Thr Asn Gln Ala Tyr Ala Ile Ala Ser Ser Ile Val Ser Phe 195 200 205 Tyr Val Pro Leu Val Ile Met Val Phe Val Tyr Ser Arg Val Phe Gln 210 215 220 Glu Ala Lys Arg Gln Leu Gln Lys Ile Asp Lys Ser Glu Gly Arg Phe 225 230 235 240 His Val Gln Asn Leu Ser Gln Val Glu Gln Asp Gly Arg Thr Gly His 245 250 255 Gly Leu Arg Arg Ser Ser Lys Phe Cys Leu Lys Glu His Lys Ala Leu 260 265 270 Lys Thr Leu Gly Ile Ile Met Gly Thr Phe Thr Leu Cys Trp Leu Pro 275 280 285 Phe Phe Ile Val Asn Ile Val His Val Ile Gln Asp Asn Leu Ile Arg 290 295 300 Lys Glu Val Tyr Ile Leu Leu Asn Trp Ile Gly Tyr Val Asn Ser Gly 305 310 315 320 Phe Asn Pro Leu Ile Tyr Cys Arg Ser Pro Asp Phe Arg Ile Ala Phe 325 330 335 Gln Glu Leu Leu Cys Leu Arg Arg Ser Ser Leu Lys Ala Tyr Gly Asn 340 345 350 Gly Tyr Ser Ser Asn Gly Asn Thr Gly Glu Gln Ser Gly Tyr His Val 355 360 365 Glu Gln Glu Lys Glu Asn Lys Leu Leu Cys Glu Asp Leu Pro Gly Thr 370 375 380 Glu Asp Phe Val Gly His Gln Gly Thr Val Pro Ser Asp Asn Ile Asp 385 390 395 400 Ser Gln Gly Arg Asn Cys Ser Thr Asn Asp Ser Leu Leu 405 410 204PRTArtificial Sequencesynthetic peptide 20Arg Thr Val Trp 1 215PRTArtificial Sequencesynthetic peptide 21Arg Thr Glu Val Trp 1 5 226PRTArtificial Sequencesynthetic peptide 22Arg Thr Asp Glu Val Trp 1 5 237PRTArtificial Sequencesynthetic peptide 23Arg Thr Ala Asp Glu Val Trp 1 5 248PRTArtificial Sequencesynthetic peptide 24Arg Thr Ala Ala Asp Glu Val Trp 1 5 259PRTArtificial Sequencesynthetic peptide 25Arg Thr Ala Ala Ala Asp Glu Val Trp 1 5 2612PRTArtificial Sequencesynthetic peptide 26Arg Thr Gly Thr Trp Asp Ala Tyr Asp Glu Val Trp 1 5 10 2713PRTArtificial Sequencesynthetic peptide 27Arg Thr Gly Thr Trp Asp Ala Tyr Ala Asp Glu Val Trp 1 5 10 2814PRTArtificial Sequencesynthetic peptide 28Arg Thr Gly Thr Trp Asp Ala Tyr Ala Ala Asp Glu Val Trp 1 5 10 2915PRTArtificial Sequencesynthetic peptide 29Arg Thr Gly Thr Trp Asp Ala Tyr Ala Ala Ala Asp Glu Val Trp 1 5 10 15

Claims

1. A GPCR fusion protein comprising, a) a G-protein coupled receptor (GPCR); and, b) an autonomously folding stable domain; wherein said autonomously folding stable domain is N-terminal to said GPCR and is heterologous to said GPCR; and wherein said GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions.

2. The GPCR fusion protein of claim 1, further comprising an epitope tag N-terminal to said autonomously folding stable domain.

3. The GPCR fusion protein of claim 2, further comprising a protease cleavage site between said epitope tag and said autonomously folding stable domain

4. The GPCR fusion protein of claim 1, wherein said autonomously folding stable domain comprises the amino acid sequence of lysozyme.

5. The GPCR fusion protein of claim 1, wherein said GPCR comprises a second autonomously folding stable domain between the TM5 and TM6 regions of said GPCR.

6. The GPCR fusion protein of claim 1, wherein said GPCR is active.

7. The GPCR fusion protein of claim 1, wherein said GPCR is naturally occurring.

8. The GPCR fusion protein of claim 1, wherein said GPCR is non-naturally occurring.

9. A composition of matter comprising: a) a GPCR fusion protein of claim 1; and b) a moiety complexed with said GPCR fusion protein.

10. The composition of claim 9, wherein said moiety complexed with said GPCR fusion protein is a G-protein.

11. The composition of claim 9, wherein said moiety is an antibody that is bound to the IC3 loop of said GPCR.

12. The composition of claim 9, wherein said moiety is a ligand for said GPCR.

13. A nucleic acid encoding the fusion protein of claim 1.

14. The nucleic acid of claim 13, wherein said nucleic acid encodes, from 5' to 3': a) a signal sequence; b) an epitope tag; c) a protease cleavage site; d) an autonomously folding stable domain; and e) a GPCR.

15. A crystal comprising the GPCR fusion protein of claim 1.

16. The crystal of claim 15, further comprising a G protein complexed with said GPCR.

17-19. (canceled)

20. A method comprising: culturing the cell of claim 13 to produce said GPCR fusion protein; and isolating said fusion protein from said cell.

21. The method of claim 20, further comprising: crystallizing said GPCR fusion protein to make crystals.

22. The method of claim 20, further comprising: obtaining atomic coordinates of said fusion protein from said crystal.

23. A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising: a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR fusion protein of claim 1 to X-ray diffraction analysis; b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least the ligand binding site of the GPCR; c) displaying on the computer system a model of said ligand binding site.